Proteins 2024 Lecture Notes (BIOC 192) PDF

LECTURE NOTES I 2024 Proteins (Lectures 2-14) Foundations of Biochemistry BIOC 192 2024 This booklet covers the Proteins section of BIOC 192 (Lectures 2-14). The booklet is designed to accompany the lectures, which will be delivered by the academic teaching staff of the Biochemistry Department. It...

LECTURE NOTES I 2024 Proteins (Lectures 2-14) Foundations of Biochemistry BIOC 192 2024 This booklet covers the Proteins section of BIOC 192 (Lectures 2-14). The booklet is designed to accompany the lectures, which will be delivered by the academic teaching staff of the Biochemistry Department. It is not a stand-alone summary of the lectures, but is designed to complement your own notes and to aid your comprehension and study. It includes figures and tables which may be shown in lectures but which would take too long to copy down. References are also given to appropriate sections of the textbook (Biochemistry and Molecular Biology, 6th edition by D. Papachristodoulou, A Snape, W.H. Elliot and D.C. Elliot). A separate document on Blackboard, entitled “recommended readings” also gives textbook references to a previously used textbook (Biochemistry 9th edition by M.K. Campbell, S.O. Farrell and O.M. Mc Dougal). You should check the BIOC 192 Blackboard website regularly to find out news and information about the paper as well as material that we hope will be of interest to you. Importantly from an examination point of view, the learning objectives for each lecture are listed in the booklet. These should be the focal points for your study. Initially you should concentrate on ensuring that you understand each lecture topic or subtopic well enough to be able to explain it in your own words to a friend. To achieve this you need to read and understand your own lecture notes and the appropriate pages of this booklet, using the textbook to aid in your understanding. Whilst recall of information from individual lectures is important, as you progress through the paper you should begin to appreciate how the various aspects of the discipline are integrated. Past exams can be accessed through the library website. Both factual recall and the ability to explain and describe aspects of biochemistry are important. Copyright Warning Notice This material may be used only for the institution’s educational purposes. It includes extracts of copyright works copied under copyright licences. You may not copy or distribute any part of this material to any other person. Where this material is provided to you in electronic format you may only print from it for your own use. You may not make a further copy for any other purpose. Failure to comply with the terms of this warning may expose you to legal action for copyright infringement and/ or disciplinary action by the institution. The lecturers for the Proteins section are Professor Peter Dearden, Professor Kurt Krause, Professor Peter Mace and Professor Debbie Hay. Part 1 Lecture Titles ”Lecture 1 & 2” Introduction to Biochemistry “Lecture 3” Proteins and cellular function “Lecture 4” Building blocks of proteins: amino acids “Lecture 5” Elements of protein structure “Lecture 6” Folding a protein “Lecture 7” Enzymes are essential for life “Lecture 8” How do enzymes catalyse reactions? “Lecture 9” Measuring and comparing the activities of enzymes “Lecture 10” Control of enzyme activity “Lecture 11 & 12” Proteins in action: Oxygen transport and storage by haemoglobin and myoglobin #1 and #2 “Lectures 13 & 14” Activation and inhibition of proteins #1 and #2 PowerPoint presentations will be made available on Blackboard (beforehand if possible). A full recommended readings list is also available on BlackBoard. Remember, if you have any problems studying this topic ask your laboratory supervisor, ask a question on discussion board (use the search function first to ensure your question has not already been asked and answered!) or come to the BIOC 192 Office (Room 132, 1st floor Microbiology), phone 479-8620, email: [email protected]. There is always help available. 1 Lecture 1 & 2 Introduction to biochemistry What is biochemistry (in context)? Molecular interactions in biochemistry Solving biochemical problems Learning Objectives At the end of your study relating to this topic you should be able: To appreciate that the structure and function of biological molecules are inextricably connected. To understand that regulation of cellular function depends on interaction of molecules, and that this in turn relies on chemical and structural complementarity. To have an appreciation of the types of questions biochemists ask. Textbook references: Chapter 1 “The basic molecular themes of life”, page 3 onwards, in Biochemistry and Molecular Biology, 6th edition by D. Papachristodoulou, A Snape, W.H. Elliot and D.C. Elliot. This chapter provides a general introduction to biochemistry, and how it intersects with biology in general, cell biology, chemistry, and physics. It is this concept we want you to focus on, rather than learning the scientific principles outlined in this chapter (a lot of this content will be covered later in the semester). 2 Lecture two aims to give a broad overview of biochemistry, highlighting some key concepts that will be covered in BIOC192. As biochemists we are interested in how molecules and molecular interactions allow cells to solve problems. We are also interested in how we can manipulate interactions – this is how many drugs function. Biochemistry focuses on the molecular basis of cellular function. Biochemists are interested in areas such as: Molecules that make up cells, how they interact, and how they function. How the cellular blueprint (genes in DNA) are regulated to underpin the development of complex multicellular organisms. How each cell type, with the same DNA, looks and functions differently. How cells access energy in food and convert it to a form that is available to drive our cellular processes. In brief, proteins are the workhorses of our cells. They are polymers consisting of amino acids joined together via peptide bonds. The specific sequence of amino acids both determines how proteins fold into their three-dimensional structure, and the function of the resulting protein. This will be covered in detail by Professor Kurt Krause Figure 2.1. The building blocks of proteins. Proteins consist of a sequence of amino acids, which fold into local structures called secondary structure, which then forms an overall 3D structure, the tertiary structure. Multiple proteins may associate into a quaternary structure. 3 Biochemists can make and isolate proteins; to aid in the investigation of cellular processes, or to produce therapeutic proteins. This will be covered in the biotechnology lectures by Professor Stephanie Hughes. Figure 2.2. Overview of the generation of recombinant proteins. First, we must find the genetic code for the protein of interest and insert it into a plasmid. Secondly, we insert the gene into a cell, the cell has the new gene inside so will produce protein, and can be made to export from cell into growth medium. The cells may be grown in stirred tanks (bioreactors) providing suitable environment with enough nutrients. Finally, the protein is harvested from ‘cell soup’. A very important question to consider is how our cells obtain energy from food, and then how do we use that energy? In plants, energy from the sun is converted by photosynthesis into the energy of chemical bonds. In animals, cells can release the energy stored in their food molecules through a series of oxidation reactions (chemical reactions in which electrons are transferred from one molecule to another), changing the composition and energy content of both the donor and acceptor molecules. The key product of these reactions is ATP – and in the second part of BIOC 192 you will learn a lot from Associate Professor Lynette Brownfield and Professor Sally McCormick about the processes that release ATP. 4 Lecture 3 Proteins and cellular function Learning Outcomes At the end of your study relating to this topic you should be able to: Describe the diversity of protein function and structure Explain the overall makeup of proteins Explain some of the key functions of proteins in the cell Provide one example of a protein involved in each of: immune defense, digestion and metabolism, DNA and RNA replication, oxygen transport Textbook references: Chapter 1 “Basic molecular themes of life” page 14 – 15, “Proteins” in Biochemistry and Molecular Biology, 6th edition by D. Papachristodoulou, A Snape, W.H. Elliot and D.C. Elliot 5 What are proteins? Amino acids COO- C-terminus NH NH3+ - H - C - COO N-terminus - R Peptide bonds Figure 3-1. The primary structure of a protein. Proteins are large biomolecules, known as macromolecules, consisting of amino acids linked together by covalent peptide bonds, i.e. they are polymers of amino acids. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their genes. There are 20 different amino acids used to make proteins. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, responding to stimuli, providing structure to cells and our physical makeup (making up our muscle, ligaments, tendons, hair, and nails!), and transporting molecules from one location to another. Amino acids Peptide Protein Figure 3-2. Polymerised amino acids fold into a protein. Why study the structure of proteins? As proteins carry out or catalyse almost all of the vitally important processes that keep our bodies alive, it is important that we understand how proteins work, and how they work together. Knowing the structure of a protein is important because the structure of a protein determines its function. Similar to the structure that we call a ‘chair’; it has four legs, a seat and a back rest. We use this ‘chair structure’ to sit on, not to e.g. eat from. Although there are many types of chairs, they all have the same basic structure, and thus have the same function. The same applies to proteins. There are a number of ways in which the structure of a protein can be determined. The most common method is called “protein crystallography” (see box below). Other commonly used methods to determine protein structure include electron cryo-microscopy (which works particularly well for very large proteins), and NMR spectroscopy. 6  Our knowledge of protein structure/architecture has come principally from X-ray crystallography. The protein of interest is isolated and protein crystals are grown. X-rays are shot at the protein crystal and are scattered by the electrons in the protein molecules making up the crystal. The scattered waves recombine to form a diffraction pattern that can be recorded on an X-ray sensitive surface. The pattern depends on the atomic arrangement in the crystal and may be converted mathematically to an electron density map from which the molecular model is built. Improvements in nuclear magnetic resonance spectroscopy (NMR have allowed the structures of small proteins to be determined. Figure 3-3. Schematic of the X-ray crystallography technique (left, Whitford, p.350) and an X-ray diffraction photograph of a glutathione synthetase crystal (right, Campbell and Farrell 7th ed, p.96). 7 Figure 3-4. Examples of actual protein structures. These structures, which have all been experimentally determined, help to illustrate the huge range in size and structure of various proteins found in the human body. Figure adapted from PDB Molecular Machinery poster. Some key functions of proteins in our cells Below are listed some examples of important functions in our bodies where proteins play a vital role. For each of these functions, one example is listed. However, as you will learn throughout this course, each of these important functions is actually mediated by a large number of different proteins each playing their particular role in the overall process. Cell signalling – e.g., insulin Digestion – e.g., trypsin, amylase Metabolism – e.g., hexokinase, alcohol dehydrogenase Oxygen transport – e.g., haemoglobin Immune protection – e.g., antibodies Energetics – e.g., ATP synthase Replication and maintenance – e.g., DNA polymerase, RNA polymerase 8 Lecture 4 Building blocks of proteins: amino acids. Learning Objectives At the end of your study relating to this topic you should be able to: Describe the properties of amino acids and how they relate to protein structure and function Recognize the types of side chains of amino acids and understand their chemical properties Know how amino acids join to form peptides and proteins Explain the importance of the peptide bond and describe its structure and key properties Textbook references: Chapter 4 “The structure of proteins” pages 51-55 in Biochemistry and Molecular Biology, 6th edition by D. Papachristodoulou, A Snape, W.H. Elliot and D.C. Elliot 9 Twenty different amino acids are the building blocks of proteins Amino acids, as the name implies, contain an amino (H2N–) group and a carboxyl (–COOH) group (these are termed the α-amino and α-carboxyl groups respectively). In a free amino acid these functional groups can ionise in solution to form carboxylate (-COO-) and ammonium (H3N+-) groups. Amino acids differ in their physical properties as a result of the structures of their side-chains (R-groups). All of these groups are linked to a central (α)-carbon atom in the amino acid. The functional groups linked to the α-carbon atom create a point of asymmetry in the amino acid and therefore stereoisomer (mirror image) D- and L- forms of amino acids exist. Living organisms make proteins that generally contain only L-amino acids. Bacteria, however, have the ability to chemically synthesise polymers that also contain D-amino acids. Figure 4-1. The general form of an amino acid Figure 4-2. Stereochemistry of an amino acid. L-configuration amino acids are found in gene- encoded proteins (Campbell, Farrell and McDougal p.61). 10 Amino acids are classified according to side chain structure The 20 amino acids commonly found in proteins differ from each other in their R-group side-chain chemical structure (see Figure 4-3). Several amino acids are classified as non-polar and have aliphatic carbon or aromatic ring side-chains which tend to be located away from aqueous environments. Other amino acids have polar side-chains and include those amino acids with ionisable acidic or basic groups. Note: the cysteine -SH is slightly polar, and could also be included with the uncharged polar group below (different texts classify it differently). Note: because in the structures above, the carboxyl group is Note: the Lys, Arg, and His side-chains are deprotonated (COO-, as would be expected at pH7), what you see shown in their protonated conjugate acid is the ionic form of the amino acids. These ionic forms are called form, as you would expect at pH 7. aspartate and glutamate. Figure 4-3. Structures of the 20 common amino acids found in proteins, shown in their form at pH 7. Some amino acids have ionisable groups that contribute to the net charge on a protein 11 Figure 4-4. Ionisation of amino acids (Campbell, Farrell and McDougal p.68). In addition to the α-amino and α-carboxyl groups, some amino acids have amino or carboxyl groups in the side-chain R-group, which can ionise and contribute to the net charge of the amino acid. The pKa value of the ionising group depends on both its location in the amino acid and on the nature of the group. In a protein, where the amino acids are covalently linked together, the net charge on the protein is derived principally from ionisation of side-chain amino and carboxyl groups. The isoelectric point (pI) of a protein is the pH at which the protein has a net charge of zero. From Table 4-1 you can see that almost all of the α-amino groups have a pKa between 9-10 and the α-carboxyl groups between 2-3. Additionally, seven other amino acids have side chains that are ionisable. Apart from Arg, these amino acids often appear in the active sites of enzymes. Remember from CHEM191: The pKa value for an ionisable group (on an amino acid in this case), is the pH at which 50% of the ionisable groups in the solution are ionised (and the other 50% are un- ionised), giving an overall net charge or zero (neutral). Table 4-1. pKa values of the ionisable functional groups of amino acids (Campbell, Farrell and McDougal p.69). 12 Amino acids are covalently linked by peptide bonds in proteins Figure 4-5. Formation of a peptide bond (Campbell and Farrell 4th ed. p.74). The C–N peptide bond linkage has partial double bond character due to electron resonance, resulting in the peptide bond being planar. The order of covalently linked amino acids in the polymer is known as the primary sequence. The amino acid with the unlinked α-amino group is known as the N-terminus of the protein (i.e. the first amino acid of the protein chain), the other end of the primary sequence containing the unlinked α-carboxyl group is known as the C-terminus (i.e. the last amino acid of the protein chain). By convention, the amino acid sequence is written N-terminus to C-terminus. This linear sequence of amino acids helps to define the structure and function of the mature protein. Individual amino acids in a protein may also be referred to as “residues”. In addition to being planar, the peptide bond is most often in a trans conformation (except for residues preceding a proline (about 10% cis)), and it has a fixed dipole. The prefixes ‘cis’ and ‘trans’ are from Latin, meaning ‘this side of’ and ‘the other side of’ respectively. In the context of (bio)chemistry, cis indicates that the functional groups (side chain R groups in the case of proteins), are on the same side of the carbon chain (the peptide bond in the case of proteins), while trans configuration means that the functional groups are on the opposing side of the carbon chain. Figure 4-6. Configuration of a peptide bond (Campbell, Farrell and McDougal p.72). Figure 4-7. Primary structure of a small peptide (Campbell, Farrell and McDougal p.71). 13 Proteins undergo post-translational modification Proteins transferred from the endoplasmic reticulum through the Golgi apparatus undergo a number of modifications, termed post-translational modifications, i.e. modifications that take place after the protein has been translated. These modifications are numerous and could include, for example, one or more of the following: proteolytic cleavage of a precursor form of the protein N-glycosylation addition of lipid formation of disulphide bridges (secreted proteins) hydroxylation (e.g. collagen) C-terminal amidation (bioactive peptides) phosphorylation (e.g. milk proteins) Modifications of this type are in most cases important for generating the correct active form of the protein. In addition to post-translational modification of newly synthesised proteins, proteins are subjected to a number of age-related modifications, for example glycation of haemoglobin, free radical damage to proteins, etc. The ultimate function of a protein is dictated by the amino acid sequence, the higher order structure of the protein, and the involvement of post-translational modifications. Protein function is quite diverse as illustrated by the following groupings: Enzymes Regulatory proteins Transport Storage Contractile & motile Structural Scaffold Protective 14 Lecture 5 Elements of protein structure Learning Objectives At the end of your study relating to this topic you should be able to: Define primary, secondary, tertiary and quaternary levels of protein structure Define the properties of the α-helix, β-sheet, and turns, and recognize them in protein structures. Understand the importance of the extent and limitations of protein bond flexibility in protein and peptide structure. Understand the concept that the ultimate function of a protein is dictated by its amino acid sequence and higher order structure Textbook references: Chapter 4 “The structure of proteins” pages 56-61in Biochemistry and Molecular Biology, 6th edition by D. Papachristodoulou, A Snape, W.H. Elliot and D.C. Elliot 15 A protein structure can be described at four levels: primary structure: the order in which amino acids are linked via peptide bonds. secondary structure: local regular folding stabilised by hydrogen bonds between backbone peptide groups e.g. α-helix, β-sheet, turns. tertiary structure: the three-dimensional arrangement of a complete polypeptide chain. quaternary structure: the way in which two or more polypeptide chains associate in a multi- subunit protein. Note that the primary structure determines all the higher levels of structure and It is the specific 3-dimensional structure of a protein that allows it to function in its particular biological role. Figure 5-1. The four levels of protein structure. (Branden & Tooze, 1999) 16 Secondary structure These are regions of regular polypeptide backbone folding due to favoured rotations of two of the three bonds in the backbone (the other bond is the rigid peptide bond). Figure 5-2. Rotation of peptide bond planes around an α-carbon (Branden and Tooze). The two most energetically favourable secondary structures are the α-helix and β-sheet (two types: parallel and antiparallel) which were originally predicted by Linus Pauling (dual Nobel Prize winner) in the 1950s. 17 The α-helix The α-helix, with 3.6 residues per turn and a pitch of 5.4 Å (Å = Ångström, a unit of length used to measure very small distances, where 1 Å = 10-10 m, i.e. 0.1 nm), optimises hydrogen bonding between the carbonyl oxygen of residue n and the amide –NH of residue (n + 4). These bonds point approximately parallel to the helix axis. The sidechains point out from the helix axis. Figure 5-3. Three representations of an α-helix showing the atomic constituents (above left and below), backbone hydrogen bonding (middle) and the configuration of side-chains along the long axis of the helix (right) (see Campbell and Farrell (4th ed), p.90; Campbell and Farrell (88h ed), p.78). Proteins have varying amounts of α-helix. For example, haemoglobin has about 75% helix whereas antibodies (immunoglobulins) have none. The length of a single helix can vary from one turn to over a hundred turns, the average being between 3 and 5 turns. The presence of some amino acids does not favour α-helix formation – glycine is too flexible, and proline cannot form a hydrogen bond with its α-amino nitrogen, and the rotation around its Cα-N bond is restricted. Certain sidechains may clash sterically or repel each other and thereby prevent α-helices from forming. 18 The β-sheet The peptide backbone in the β-pleated sheet (β-sheet for short) is extended and the hydrogen bonds are formed between different parts of a single polypeptide chain running in either a parallel or antiparallel direction. Viewed side-on the sheet has a zigzag (pleated) structure with the amino acid sidechains pointing alternately up and down. The sheet is not flat but has a slight right-handed twist. Figure 5-4. Two representations of a β-sheet showing the configuration of side-chains along the backbone of the sheet strands (top) and the arrangement and hydrogen bonding in parallel (bottom left) and anti-parallel (bottom right) β-sheets (see Campbell and Farrell (4th ed), p.91; Campbell, Farrell and McDougal (9th ed), p.81). Each vertical ‘column’ in the bottom figure corresponds to a β-strand. Proteins contain variable amounts of β-sheet and there are, as with the α-helixes, constraints on which amino acids are tolerated. The size and charge of sidechains is an even more important consideration in β-sheet structures, and once again proline is unwelcome! When there is just one chain in this conformation (or when we are talking about just one part of the β-sheet), we often refer to it as a β-strand. When there are multiple chains, we then use the term β-sheet. You could think of it this way: a β-sheet is made of multiple β-strands. 19 Other elements of secondary structure α-helices and β-sheets are not the only types of secondary structure found in proteins. There are other types of helices and also loops that are found in proteins. Another group of important secondary structure elements are the turns. As their name suggests, turns allow polypeptide chains to change direction. They are often found between strands of anti-parallel β-sheet (hence they are often called β-turns). There are a variety of different types of turns, but commonly they consist of four amino acids, with the second one (from the N-terminal end) often being proline. Because the side-chain of proline connects back onto the α-amino group of the amino acid (see Figure 5-2), it introduces a natural bend in a polypeptide chain that is useful for changing direction. Figure 5-5. Examples of β-turns (see Campbell, Farrell and McDougal, p.83). Main chain bond angles From looking at Figure 5-2, it is easy to get the impression that there is free rotation of the bonds on either side of an α-carbon in a polypeptide. However, what is not taken into account in this diagram is that for some rotations of φ (phi) and ψ (psi), the side-chains of two amino acids in a polypeptide would collide. Expressed more formally, the side-chains would approach each other within their van der Waals radii and this type of interaction is sterically blocked. In fact, this type of steric interference occurs for most angles of phi and psi. 20 Lecture 6 Folding a protein Learning Objectives At the end of your study relating to this topic you should be able to: Describe the interactions that stabilise the tertiary structure of a protein. Demonstrate that most proteins contain domains of helices and sheets that make up the tertiary structure. Understand the concept that the ultimate function of a protein is dictated by its amino acid sequence. Describe Anfinsen’s experiment showing this. Outline the steps involved in folding a newly synthesized protein, including the role of chaperones. Describe how a misfolded protein can lead to disease and provide an example. Textbook references: Chapter 25 pages 410-413 from “folding up of the polypeptide chain” up to, and including “protein folding and prion disease” in Biochemistry and Molecular Biology, 6th edition by D. Papachristodoulou, A Snape, W.H. Elliot and D.C. Elliot 21 The elements of secondary structure discussed in Lecture 5 can be combined in different ways to build up step by step the structure of a protein. Simple combinations of strands and helices can be used to form supersecondary structures. Supersecondary structures form domains. Mature proteins are comprised of one or more domains. Supersecondary structure Secondary structure elements are connected (usually) by structured loops. Supersecondary structures are combinations of secondary structures that form recognisable patterns, e.g. αα, ββ, βαβ, ββββ (Figure 6-1). These in turn build up into tertiary structure. Figure 6-1. Examples of supersecondary structures (Campbell, Farrell and McDougal, p.84) Tertiary structure This refers to the 3-dimensional structure of a protein, i.e. the arrangement of all of its atoms in space. Globular proteins, including enzymes, transport proteins and immunoglobulins are compact folded structures (compared to the extended structures of fibrous proteins) that have extensive regions of secondary structure. In contrast to the short-range interactions (mainly hydrogen bonds) of secondary structure, tertiary structure is stabilised by long range interactions involving amino acid residues that may be far apart in the primary sequence. The structure is maintained by non-covalent interactions, and in the case of extracellular proteins often by covalent disulphide bonds (e.g. insulin). These are formed by the oxidation of two cysteine sidechains in close proximity. Hydrophobic sidechains cluster on the inside of the protein away from solvent, while polar (hydrophilic) sidechains are on the outside. Hydrogen bonds may involve backbone (non-α-helix or β-sheet regions) or sidechain atoms. Ionic bonds involve just the ionisable sidechains and the two termini, and are much less common. Sometimes a metal ion such as Zn2+ or Ca2+ is bound, adding stability to the protein. Figure 6-2. The various bonds that stabilise tertiary structure (Campbell, Farrell and McDougal, p.88). 22 Protein domains A domain is a relatively stable independently folded region within the tertiary structure of a globular protein. A protein might contain one, two or more domains, which often have a particular function associated with them (such as a ligand binding function). There are many different protein structures but we now recognise ‘families’ whose members have similar structures even though their primary structures may be quite different. Ribbon presentations of structures are convenient for identifying and comparing proteins. Figure 6-3. Examples of the amount of secondary structure found in different families/domains of tertiary structure (Mathews, van Holde and Ahern, p.179). They are shown using ribbon diagrams, in which a ribbon is shown tracing the pathway of the protein main chain. Arrows show the N to C direction of β-strands. Quaternary structure Usually refers to the non-covalent interaction between two or more folded separate polypeptide chains. In quaternary structures, each tertiary structure is referred to as a subunit. These assemblies are called dimers (for 2 subunits), trimers (3 subunits), tetramers (4 subunits) etc. 23 Protein folding Protein folding is driven largely by hydrophobic forces, that is, it is the internal hydrophobic residues that direct folding to the native conformation (with its nonpolar core). The formation of α-helix and β-sheet structures occurs early in the folding process and is referred to as nucleation. The protein becomes more compact as the secondary structure elements (supersecondary structures, motifs and domains), come together to form larger folding units. The final stage involves relatively small adjustments in conformation to yield the native protein structure. The whole process follows an ordered pathway (a random search of all possible conformations would take billions of years) and is cooperative, i.e. each folding step facilitates the formation of another favourable step in the pathway. Most globular proteins will become denatured (unfolded) and consequently lose their biological function following heat treatment or exposure to detergents, organic solvents, various chemicals and extremes of pH. These treatments disrupt the many non-covalent interactions that hold the native (correctly folded) protein together. Depending on the harshness of the treatment and the particular protein involved, denaturation may be reversible. In the 1950s Christian Anfinsen (a Nobel Prize winner) carried out a renaturation experiment on the enzyme ribonuclease A, proving that the amino acid sequence of a protein contains all the information required for determining the 3-dimensional structure. Anfinsen’s experiment, in brief, involved unfolding the protein ribonuclease A (a hydrolytic enzyme that degrades RNA) in 8M urea, assisted by a thiol reagent (mercaptoethanol) to break its four disulphide bonds. Following removal of the reagents by dialysis and exposure to oxygen, some native (folded and fully active) ribonuclease A, containing the correctly paired disulphides, was slowly re-formed. We now know that in vivo, the rate of disulphide bond formation is increased with the help of an enzyme (protein disulphide isomerase). This enzyme is one of several accessory proteins that assist polypeptide folding in vivo. Without them, folding a newly synthesised polypeptide Figure 6-4. Schematic of Anfinsen’s would be slow and inefficient. experiment (Stryer, 4th ed). Thus, the primary sequence of a protein governs its 3-dimensional structure, but to fold correctly and efficiently, accessory proteins are often needed to assist the process. 24 Particularly important in assisting folding are the so-called molecular chaperones, which work by preventing improper folding. One type of chaperone, called a chaperonin, is a large multi-subunit protein that resembles a rubbish bin in appearance. Figure 6-5. Chaperone-independent (a), chaperone -mediated (b) and chaperonin assisted (c), protein folding (Campbell, Farrell and McDougal, p.373). 25 Misfolded proteins and disease There are a variety of disorders caused by misfolding and/or aggregation of proteins that become deposited in various tissues. Examples include prion diseases (see below), Alzheimer’s Disease, and Type II diabetes. In these former two disorders, it is believed that the abnormally folded protein, amyloid, contributes to pathogenesis. Prion diseases Prion diseases are a unique type of infectious disease where the agent that transmits the disease is not a living organism or virus, but instead is a protein. The prion protein (PrPC) is a normal cellular protein expressed in the central nervous system, lymphatic tissue and at neuromuscular junctions. The pathogenic form (PrPSc) is covalently the same as PrPC (has the same amino- acid sequence) but has a different conformation (increased β-sheet content), that promotes self-association into stable aggregated particles. PrPSc is resistant to digestion by proteases and forms aggregates inside infected cells, forming plaques. The abnormally folded form of the prion protein is able to cause the normal form to change its conformation to the abnormal one. Prion diseases have been characterised in cows (Bovine Spongiform Encephalopathy (BSE), a.k.a. ‘mad cow disease’), sheep (scrapie) and in humans (Creutzfeldt-Jakob disease (CJD – ‘human mad cow disease’)). As well as the transmissible form of the disease, there are genetic and sporadic forms. Figure 6-6. The normal conformation of prion protein vs. what is thought to be the pathogenic conformation of prion protein. 26 Lecture 7 Enzymes are essential for life Learning Objectives At the end of your study relating to this topic you should be able to understand and describe: The general roles and types of enzymes. Thermodynamic features of an enzyme catalyzed reaction. Roles of cofactors in enzyme-catalysed reactions. Individual enzymes do not act in isolation. Textbook references: Chapter 3 “Energy considerations in biochemistry” pages 36-39 and Chapter 6 “Enzymes” pages 95-97, 102 in Biochemistry and Molecular Biology, 6th edition by D. Papachristodoulou, A Snape, W.H. Elliot and D.C. Elliot 27 Enzymes as biological catalysts The most important function of enzymes is that of biological catalysis. Catalysts are compounds which speed up reactions (increase the rate or velocity of reactions) but do not alter the reaction equilibrium. At equilibrium the ratio of the reactant (substrate) concentrations to the product concentrations is the same, whether or not enzyme is present. The increase in reaction rate is achieved by lowering the free energy of activation of the reaction, thereby allowing the transition state to be reached more easily. For example, the enzyme catalase lowers the activation energy for the decomposition of hydrogen peroxide from 75.2 kJ∙mol-1 to 23.0 kJ∙mol-1. This results in an increase in rate of 6.5x108. Features of enzyme catalysed reactions 1. Much faster reaction rates: Enzyme catalysed reactions are much faster (by 106 - 1012 times) than the corresponding uncatalyzed reaction and many times faster than chemically catalysed reactions. 2. Much milder reaction conditions: Enzyme catalysed reactions are efficient at the mild conditions of temperature and pH found inside biological cells. In contrast, chemically catalysed or uncatalyzed reactions often require high temperatures and extremes in pH and/or pressure. 3. Reaction specificity: Enzymes are specific to their substrates (reactants). This is useful in intracellular conditions where hundreds and thousands of different substrates and reactions coexist. 4. Tightly regulated: There are many mechanisms that ensure the tight control of enzyme catalysed reactions, ensuring that their activity is efficient when and where required. Energy profiles of uncatalysed and catalysed reactions Figure 7-1. Activation energy profiles (Campbell, Farrell and McDougal, p.142). (a) The activation energy profile for a typical reaction. The reaction shown here is exergonic (energy-releasing). Note the difference between the activation energy (∆G°‡) and the standard free energy of the reaction (∆G°). (b) A comparison of activation energy profiles for catalysed and uncatalysed reactions. The activation energy of the catalysed reaction is much less than that of the uncatalysed reaction. 28 Enzyme nomenclature The name of an enzyme is usually indicative of the type of reaction catalysed by that enzyme. Many enzymes were named by adding the suffix –ase to the name of their substrate. For example, urease catalyses the hydrolysis of urea. But we also come across other names, like trypsin and pepsin, that do not denote their substrates. Sometimes enzymes have been given different names at different times, adding to the confusion. In the 1960s, an international Enzyme Commission was set up to standardise the classification of enzymes. This system places all enzymes in six major classes (with subclasses), based on the type of reaction they catalyse. Although it might seem perverse to go to all this trouble to classify enzymes, this has avoided a lot of ambiguities. In addition, if the proper (systematic) name for an enzyme is long and cumbersome, a short name may be used. Most enzymes catalyse the transfer of electrons, atoms or functional groups. They are therefore classified, given code numbers and assigned names according to the type of transfer reaction, the group donor and the group acceptor. Table 7-1. International classification of enzymes, based on the reactions they catalyse. Catalytic mechanisms The enzyme participates in the chemistry of the reaction through its catalytic groups, which are amino acid sidechains that can act as acids, bases, nucleophiles and electrophiles. Enzymes undergo numerous types of catalytic mechanisms, including redox reactions, the use of cofactors, and many more. Two important types of catalytic mechanism are discussed briefly below. Both covalent catalysis and acid-base catalysis will be discussed further in later lectures. Covalent catalysis is one type of catalytic mechanism that some enzymes employ. A covalent enzyme- substrate intermediate is briefly formed. It is highly reactive or unstable and has a high probability of entering the transition state and completing the reaction. The serine proteases – a group of enzymes that have a reactive serine in the active site – use covalent catalysis. The digestive enzymes trypsin and chymotrypsin are both serine proteases. Other enzymes also use a covalent bond involving serine, cysteine or lysine. Acid-base catalysis involves the donation or acceptance of a proton by acidic or basic groups within the protein. This mechanism can function instead of or together with covalent catalysis. Groups acting as general acids/bases are the sidechains of Glu, Asp,Lys, Arg, His and Cys. This type of catalysis is very common. 29 Reaction Coupling – How do we ensure unfavourable, but vital, reactions occur? The Gibbs free energy of a reaction tells us about the thermodynamics of a reaction and if it is going to be energetically favourable or not. If ΔG < 0 reaction is spontaneous/ energetically favourable If ΔG = 0 the reaction is at equilibrium (uncommon) If ΔG > 0 the reaction is not spontaneous/energetically unfavourable (the reverse reaction will be favourable). If a reaction is unfavourable, then this can be coupled to an energetically favourable reaction to allow the unfavourable one to proceed. This is usually made possible by enzymes. Remember, that enzymes cannot lower the free energy of a reaction, but they can ensure that coupling does occur. The process of coupling is very common in metabolic pathways, such as the energy investment phase of glycolysis, where hexokinase catalyses the reaction: Glucose + phosphate glucose-6-phosphate + H2O This reaction has the unfavourable ΔG of +14 kJ/mol. But it is vitally important. Without this reaction, we would not be able to process glucose as a fuel source. To ensure that this reaction proceeds, it is coupled to ATP hydrolysis (ΔG -30 kJ/mol) to give the coupled reaction an overall ΔG of -16 kJ/mol, therefore making it energetically favourable. Therefore, through the action of hexokinase: Glucose + phosphate + ATP glucose-6-phosphate + ADP + H2O. Cofactors Cofactors are derived from minerals and thus, they are small, inorganic molecules. Metal ions commonly function as cofactors; recall Fe2+ and its role in oxygen binding in haemoglobin. In this instance, the Fe2+ is acting as a vital cofactor required for oxygen transport by haemoglobin. Many enzymes require cofactors. Without these cofactors, the enzymes are unable to function. Metal ion cofactors participate in the catalytic process but remain themselves unchanged in the process. A metal ion in the active site of an enzyme may play any one of numerous different roles. Some common roles of cofactors include aiding in the formation of a nucleophile or electrophile, promote binding of the enzyme to its substrate by acting as a bridge to increase binding energy, participate in charge stabilisation, or transition state stabilisation. 30 Lecture 8 How do enzymes catalyse reactions? Learning Objectives At the end of your study relating to this topic you should be able to understand and describe: The features of enzyme active sites and how substrates interact with them. Enzyme structures are dynamic and catalysis typically involving movement. Transition state analogues as drugs. The main ways that an enzyme can reduce ΔG to accelerate a reaction. Textbook references: Chapter 6 “Enzymes” 102-109 in Biochemistry and Molecular Biology, 6th edition by D. Papachristodoulou, A Snape, W.H. Elliot and D.C. Elliot 31 Active site stereospecificity Enzymes display both geometrical specificity and stereospecificity. Enzymes are specific in binding chiral substrates and in catalysing their reactions and bind their substrates through several weak bonds. This is important as the multiple weeks bonds provide specificity and allows the resulting product(s) to be released. By virtue of their chirality (proteins consist of L-amino acids), enzymes have asymmetric binding sites. A substrate of the wrong chirality will not fit into an enzyme binding site (for much the same reason as you cannot fit a right hand into a left glove or a right foot into a left shoe). In addition to this stereospecificity, enzymes are quite selective about the shape of their substrates, but most catalyse the reactions of chemical compounds that are similar in shape or size to their favourite substrate. For example, yeast alcohol dehydrogenase (YADH) catalyses the oxidation of small alcohols reasonably well, but not as well as its favourite alcohol, ethanol. Methanol and propanol, which differ from ethanol by the deletion or addition of a single CH2 group, are oxidised by YADH at a rate of 25 times and 2.5 times slower respectively, than ethanol. Figure 8-1. Enzymes show geometric and stereospecificity (Voet, Voet, and Pratt, Biochemistry). 32 Enzyme-substrate complex at the active site The first step in an enzyme catalysed reaction is the formation of an enzyme-substrate complex. There may be one or more substrates bound to the enzyme. The formation of this complex leads to the transition state species, which then forms the product(s). The enzyme binds the substrate(s) in a region of the enzyme called the active site. However, for the amino acids side chains in the active site to be able to react with its substrate, they must be close together and in the right orientation to each other. The main features of an active site are: It takes up a relatively small portion of the total volume of enzyme. It is a 3-dimensional entity. Binding of substrate can be a direct fit or an induced fit (see below). Most substrates are bound to enzymes by relatively weak forces. Active sites are clefts or crevices. The active site cleft is formed by folding the polypeptide chain into a specific shape. The parts of the protein not directly involved in the active site are needed to stabilise the tertiary structure. The lock and key hypothesis of Fischer explains enzyme specificity but it does not really help us understand catalysis. Koshland realised that an enzyme does not simply accept its substrate, it must also distort it into something close to the transition state and his induced fit hypothesis is still the dominant model for enzymatic catalysis. Experimental evidence for the model has come from X-ray crystallography and spectroscopic studies. Once the substrate is bound and the transition state is formed, the bonds are rearranged. This happens with the assistance of certain amino acid sidechains that form the active site. Bonds are broken, new bonds are formed, and the substrate is transformed into the product(s). Once the product is released, the enzyme is free to catalyse the reaction of new substrate to product. Figure 8-2. Two models for enzyme-substrate interaction (Mathews, p.369) (a) The lock-and-key model. (b) The induced fit model. In this elaboration of the lock-and-key model, both enzyme and substrate are distorted on binding. The substrate is forced into a conformation approximating the transition state; the enzyme keeps the substrate under stress. 33 Induced fit between glucose and hexokinase Hexokinase undergoes an induced fit conformational change when glucose binds, preventing the hydrolysis of ATP. Hexokinase has two conformational states: 1. The open state occurs prior to glucose binding. ATP is bound to the large lobe of the enzyme. This is far away from the glucose binding site. 2. The closed state occurs once glucose binds to the hexokinase active site. This change closes the two lobes around the glucose substrate. How does an enzyme lower ΔG‡? Enzymes have the important task of lowering the amount of energy that needs to be put into a reaction to make it proceed – that is, they lower the activation energy (ΔG‡). Don’t forget that enzymes don’t change a reaction’s ∆G value. They do not affect the free energy of the reactants or products. This means that an enzyme does not alter whether a reaction is energy-releasing or energy-absorbing overall Instead, enzymes lower the energy of the transition state, an unstable state that products must pass through to become reactants. The transition state is at the top of the energy “hill” in figure10-3. Enzymes achieve their goal in lowering the activation energy of a reaction via several mechanisms. These include: 1. Ground state destabilization – this concept suggests that there is an increase in energy in going from one state to another. Therefore, when a substrate binds to the active site of Figure 8-3 an enzyme, there is a destabilization of the substrate. This acts to reduce the activation energy of the reaction. 2. Transition state stabilisation - In binding the substrate to the active site, enzymes can stabilize the structure of the transition state. This lowers the free energy of the transition state. The more stable the transition state, the faster the reaction will be. 34 Catalytic mechanisms Enzymes employ several different catalytic strategies to enhance reaction rates. 1. Preferential binding of the transition state – by preferentially binding to and stabilizing the structure of the transition state, the reaction can proceed at an appropriate pace. 2. Proximity and orientation effects – an enzyme can mediate the interaction between two molecules by ensuring the molecules needing to react together are both close together, and in the correct orientation 3. Acid-base catalysis 4. Metal ion catalysis 5. Covalent catalysis Covalent Catalysis As the name suggests, covalent catalysis involves the formation of a transient covalent bond between the substrate and a residue in the enzyme active site (or sometimes with a cofactor at the enzyme active site). In such catalytic mechanisms, an additional covalent intermediate is added to the reaction. Usually, the covalent bonds are able to be formed as the result of an attack by a nucleophilic group on the enzyme, with an electrophilic group on the substrate. Covalent catalysis and acid-base catalysis often occur together. Figure 8-4. Overview of the concept of covalent catalysis. There are three stages in covalent catalysis: Nucleophilic reaction between the enzyme and the substrate Electrophilic withdrawal of electrons from the substrate Elimination reaction (this is the reverse of the first step) 35 Acid-base catalysis Acid-base catalysis involves the partial proton transfer from an acid to a base or vice versa. By donating or accepting electrons, the free energy of the reactions transition state in lowered. Ionisable amino acids are commonly involved in acid-base catalysis. Histidine is also particularly susceptible to acid-base catalysis as it can both accept and donate protons, depending on the pH of the environment. The hexokinase enzyme uses Mg2+ as a cofactor. Hexokinase adds a phosphate group to glucose to form glucose-6-phosphate. This is an energetically unfavourable reaction and is therefore coupled to ATP hydrolysis. When the hexokinase enzyme closes around its glucose substrate, the Mg2+ ion establishes the correct orientation/specific geometry in the active site. This ensures that the phosphate is made a better leaving group by stabilising the negative charge. Thus, Mg2+ ensures that the phosphate group will be removed from ATP, releasing the energy required for the phosphorylation of glucose. Figure 8-5. Mg2+ as cofactor in hexokinase Transition state analogues as drugs The transition state is a chemical species that is intermediate in structure between the substrate and the product. Scientists have shown, through the use of transition state analogues (molecules designed with a shape that mimics the transition state of a reaction), that enzymes stabilise the transition state, meaning that the tightest binding occurs between the transition state and the enzyme. This means that transition state analogues can make ideal enzyme inhibitors. If a transition state analogue can be designed that binds strongly to the enzyme active site, the enzyme can be inhibited, no longer being able to bind substrate. Examples of such drugs include protease inhibitors in HIV treatment. 36 Lecture 9 Measuring and comparing the activities of enzymes Learning Objectives At the end of your study relating to this topic you should be able to understand and describe: how and why to compare enzymes, by knowing the significance of KM, Vmax, kcat, and kcat/KM. how to measure these parameters using the approach of Michaelis and Menten. the range of kinetic parameters observed for enzymes, and what it can mean for biology. Textbook references: Chapter 6 “Enzymes” particularly pages 98 - 104, in your textbook Chapter 4, page 71, section entitled “theoretical models to explain protein allostery” in Biochemistry and Molecular Biology, 6th edition by D. Papachristodoulou, A Snape, W.H. Elliot and D.C. Elliot 37 Kinetics (rates of reactions) are very important for allowing the body to maintain its steady state. Reaction rates are constantly being adjusted in response to the environment and to hormonal controls. We can study the kinetics of an enzyme-catalysed reaction in the laboratory under carefully chosen conditions. The time course of an enzyme-catalysed reaction is generally complex. Enzyme reactions are usually characterised by measuring the initial rate (velocity) of the reaction V (sometimes written as V0 or Vi) which is the reaction rate at time zero. The rate depends on the available substrate concentration [S], and as substrate is converted into product (P) the rate will slow down. Figure 9-1. Time course of product formation for an enzyme catalysed reaction showing the initial rate. We calculate the initial rate of reaction by drawing a tangent to the initial, linear, section of the progress curve. The slope of this tangent represents Vo, the initial rate of reaction. The influence of enzyme concentration on the initial rate of reaction can be seen when the concentration of substrate is in excess. Under these conditions, the rate of reaction is proportional to enzyme concentration. Figure 9-2. Relationship between enzyme concentration and initial velocity under substrate saturating conditions. 38 If we measure the initial velocity at different initial substrate concentrations, we can see how the reaction rate changes in response to substrate availability. At low [S], V increases linearly with increasing [S], as in a first order reaction. At increasing substrate concentrations, the rate increases gradually. Finally, a stage is reached when there is only a vanishing increase in the reaction rate with increasing [S]. The rate is now independent of substrate concentration (zero-order kinetics). A plateau is reached, called the maximum velocity (Vmax), where the enzymes’ active sites are all occupied (saturated) by substrate. For most enzymes a graph of V against [S] is a hyperbola, and can be described by a relatively simple equation known as the Michaelis-Menten equation. The equation involves two parameters, Vmax and KM, which can be identified on a graph and which are specific for the particular enzyme-substrate pair. Figure 9-3. The effect of substrate concentration on reaction velocity (Campbell, Farrell and McDougal, p.150). Figure 9-4. Kinetic profile as a function of substrate concentration (Campbell, Farrell and McDougal, p.148). At the point where [S] = KM, the reaction proceeds at exactly half its maximum velocity. Note that it is difficult to estimate maximum velocity from a V versus [S] graph, because Vmax is approached asymptotically. We will see shortly how to estimate Vmax more accurately. The reason for this kinetic behaviour is the formation of an enzyme-substrate complex (ES). The formation of this complex is a necessary step for enzyme activity. This basic concept was expanded to a general theory in 1913 by Leonor Michaelis and Maud Menten. 39 The Michaelis-Menten approach to enzyme kinetics The enzyme first combines with the substrate(s) to form an enzyme-substrate complex, in a rapid reversible step. The ES complex then breaks down in a slower second step to give the free enzyme and the reaction product P. The second part of the reaction is slower, so limits the whole reaction. Therefore, the overall rate of the reaction depends on the concentration of the rate-limiting species ES. At any given point, the enzyme exists in two forms E and ES. At low [S] most of the enzyme will be in the free form, E. Here, the rate of the reaction will be proportional to [S]. The maximum initial rate Vmax will be observed when virtually all the enzyme is present as the ES complex, and the concentration of E is very small. Under these conditions, the enzyme is said to be saturated. After ES breaks down the enzyme is free to catalyse another reaction. The saturation effect is a distinguishing characteristic of enzyme catalysis and responsible for the shape of the kinetic curve (the plateau). The important thing to remember is that the relationship between substrate concentration [S] and enzymatic reaction rate V can be expressed quantitatively, after making the following assumptions: The rate of formation of enzyme-substrate is equal to the breakdown of the complex and it reaches a steady state very quickly. The concentration of substrate is much greater than the concentration of enzyme. The concentration of substrate does not change much during the initial stages of the reaction. In the initial stages of the reaction so little product is present that it need not be considered. This simplifies the equations shown above to: Michaelis and Menten derived the following equation: (you don’t need to know how this was derived). [S] = the substrate concentration. V = the measured initial velocity of reaction at substrate concentration. Vmax = the maximal reaction velocity measured at high concentrations of substrate. KM = the Michaelis constant: defined formally as the ratio of rate constants. 40 KM is also equivalent to the substrate concentration at which V is one-half of Vmax. The value of KM and Vmax are characteristic for each enzyme-substrate pair. What does the Michaelis-Menten equation tell us? All enzymes that have a hyperbolic relationship between V and [S] are said to follow Michaelis-Menten kinetics. Many enzymes exhibit Michaelis- Menten kinetics but do not depend on the simple two-step reaction mechanism proposed by Michaelis and Menten. However, it provides a simple language with which to compare the catalytic efficiencies of enzymes. Enzyme Substrate KM (mM) catalase H2O2 25 ATP 0.4 hexokinase (brain) D-glucose 0.05 D-fructose 1.5 Table 9-1. KM for selected enzyme-substrate pairs. KM has units of concentration. 41 The Lineweaver-Burk Plot The simplest way to calculate KM and Vmax is to plot the Michaelis-Menten graph in a different way. The Michaelis-Menten equation can be rewritten as follows (no need to memorise). This means that a plot of 1/V against 1/[S] will give a straight line, from which it is easy to determine KM from the intercept of the x-axis and Vmax from the intercept of the y-axis, as shown below: Figure 9-5. A Lineweaver-Burk plot. In this double reciprocal plot, 1/V is plotted versus 1/[S]. A linear extrapolation of the data points gives both Vmax and KM. Significance of KM and Vmax We have defined KM in two ways (with regard to Vmax and with regard to rate constants), both of which are useful. Now consider that if the ES dissociation rate constant k-1 is much larger than the rate constant for formation of product k2 (which is often the case), then KM is approximately equal to k-1/k1. This ratio is the dissociation constant for ES, so that KM is often a measure of how tightly the substrate is bound to the enzyme. The smaller the value of KM the more tightly the substrate is bound. Vmax is related to the turnover number of an enzyme, which represents the number of moles of substrate converted to product per unit time per mole of enzyme. The turnover number is usually referred to as the catalytic rate constant kcat where kcat = Vmax /[Et]. The ratio kcat / KM is a measure of an enzyme’s efficiency. It essentially describes how quickly bound substrate can be converted to product relative to how well/easily that substrate can be bound by the enzyme. A high value for the ratio indicates high efficiency in the context of overall binding and catalysis rates. 42 Lecture 10 Control of enzyme activity Learning Objectives At the end of your study relating to this topic you should be able to understand and describe: Concepts of enzyme inhibition: reversible and irreversible inhibition, and competitive and non- competitive inhibition. The predictable ways inhibition changes reaction kinetics. The range of inhibitors and activators. Textbook references: Chapter 6 “Enzymes” particularly pages 98 - 104, in your textbook Chapter 4, page 71, section entitled “Theoretical models to explain protein allostery” in Biochemistry and Molecular Biology, 6th edition by D. Papachristodoulou, A Snape, W.H. Elliot and D.C. Elliot 43 Enzyme inhibition Enzymes catalyse almost every process in a cell, so it is not surprising that the activity of many powerful poisons and drugs can be explained by enzyme inhibition. Aspirin, penicillin, nerve gases and cyanide are all enzyme inhibitors. There are two types of enzyme inhibitors: reversible and irreversible inhibitors. Reversible inhibition can be competitive or non-competitive (pure or mixed). Figure 10-1. Two types of reversible inhibition. Competitive inhibitors bind to the enzyme’s active site. Non- competitive inhibitors generally bind at a separate site. KI is the equilibrium constant for inhibitor binding (Lehninger, p.220). 44 Competitive inhibition A competitive inhibitor competes with the substrate for the active site of an enzyme, but the reaction usually does not occur when the inhibitor (I) is bound. When the inhibitor is bound to the enzyme it prevents binding of the substrate. Competitive inhibitors are usually similar in shape and chemistry to the substrate and combine with the enzyme to form an EI complex. The inhibition can be overcome by adding more substrate. When more substrate is added, the probability that the enzyme binds to inhibitor is minimised. The KM increases in the presence of inhibitor, but the Vmax remains the same. Think about why this is the case. Example Methanol is very toxic and ingestion can lead to blindness and death. Methanol is converted to the toxic compound formaldehyde by the action of the enzyme alcohol dehydrogenase (ADH). The therapy for methanol poisoning is intravenous infusion with ethanol, which competes with methanol as a substrate for ADH. This slows down the production of formaldehyde so that the methanol can be excreted harmlessly in the urine. ADH is the enzyme that processes the ingested ethanol contained in alcoholic beverages. Non-competitive inhibition The inhibitor binds at a site on the enzyme other than the active site and enzyme does not work when the inhibitor is bound. Thus, the inhibitor effectively lowers the enzyme concentration and also lowers the Vmax. Depending on where the inhibitor binds, it may or may not also affect the binding of the substrate. In pure non-competitive inhibition, the binding of the inhibitor does not affect the binding of the substrate, hence there is no effect on KM. In contrast, in mixed non-competitive inhibition, the binding of the inhibitor distorts the active site so that the substrate no longer can bind (or cannot bind so well), hence KM increases. with with with inhibitor inhibitor inhibitor 1 1 1 no inhibitor no inhibitor no inhibitor V V V Pure Mixed Competitive non-competitive non-competitive 0 1 0 1 0 1 [S] [S] [S] Figure 10-2. Lineweaver-Burk plots showing the effects on KM and Vmax in competitive inhibition, pure non- competitive inhibition and mixed non-competitive inhibition. N.B. Both competitive and non-competitive inhibitors are reversible because the inhibitors do not bind covalently to the enzyme. Irreversible enzyme inhibitors These bind covalently to enzymes and inactivate them irreversibly as they remain permanently bound to the enzyme. Many natural toxins are irreversible inhibitors of enzymes. The penicillins are irreversible inhibitors of enzymes involved in bacterial cell wall synthesis. 45 Allosteric enzymes The word allosteric comes from the Greek allos, “other” and “stereos, “shape”. This denotes the fact that allosteric enzymes change shape, or conformation, on binding of a modulator. Allosteric enzymes tend to have several protein subunits. Allosteric binding sites are distinct from the active site (as discussed previously for non-competitive inhibitors). Allosteric enzymes do not follow Michaelis- Menten kinetics. Instead, the curve of V versus [S] has a sigmoidal (rather than hyperbolic) shape. Sigmoidal kinetic behaviour usually means that there is a cooperative interaction between subunits. The binding of oxygen to the protein haemoglobin is an example of a cooperative interaction. Similarly to haemoglobin, multimeric proteins whose activity is regulated by modulators has a high (R state) and low (T state) affinity configuration for its substrate. These modulators tend to be allosteric regulatory molecules. Activators stabilise the R state. When the protein is in its R state, it already has a high affinity for its substrate. This means that the enzyme is not dependent (much) on cooperativity to increase the affinity for its substrate. Hence the curve becomes more hyperbolic and shifts to the left, almost exhibiting Michaelis-Menten enzyme behaviour. In contrast, inhibitors stabilise the T state. So, when a substrate binds to one subunit, the protein is much more dependent on cooperativity to increase the affinity for the substrate in the other subunits. For this reason, in the presence of an inhibitor, the V vs [S] curve becomes more sigmoidal and shifts to the right. Figure 10-3. a) The sigmoidal curve of an allosteric enzyme. b) The effect of an allosteric activator or inhibitor on the curve (Campbell, Farrell and McDougal, p.170). The effects that allosteric inhibitors and activators (known collectively as modulators) have on reaction velocity can be seen by looking at the relevant V vs [S] curves. The binding of an allosteric inhibitor shifts the curve to the right (and becomes more sigmoidal), while binding of an allosteric activator shifts the curve to the left (and becomes less sigmoidal/more hyperbolic). Allosteric enzymes have important regulatory roles in cell metabolism. In many metabolic processes in the cell several enzymes work in a sequential manner. The product of the first reaction becomes the substrate for the next reaction and so on. You will come across examples of several multienzyme metabolic pathways in future lectures. 46 Quite often, one of the enzymes sets the rate of the overall pathway because it catalyses the slowest (rate-limiting) step. These regulatory enzymes have increased or decreased catalytic activity in response to different signals. The activity of regulatory enzymes is controlled by some type of signal molecule, usually a small metabolite or a cofactor. Figure 12-4. Schematic of multistep pathway. Regulatory enzymes such as Enzyme 1 usually display allosteric behaviour. Allosteric regulation of glycogen phosphorylase Glycogen phosphorylase catalyses the rate limiting step in the glycogen breakdown pathway (glycogenolysis). The activity of this enzyme is regulated by both allosteric activators and inhibitors. This allows the activity of glycogen phosphorylase to be increased or reduced, depending on the requirement for glucose storage or mobilisation. Glycogen phosphorylase activity is promoted via allosteric binding of AMP. An increase in AMP concentration signals energy demand. AMP activates glycogen phosphorylase by changing its conformation from the T-state to the R-state. In the R-state, the activity of the enzyme is upregulated. This allows more glucose to be mobilised from glycogen, and therefore, energy demands can be met. High concentrations of glucose-6-phosphate suggest that glucose concentration in the cell is sufficient/in excess and therefore, glycogenolysis is not required. To ensure that glycogen is not mobilised under such conditions, glucose-6-phosphate signals feedback inhibition of glycogen phosphorylase. This acts to inactive the glycogen phosphorylase enzyme. 47 Lecture 11 & 12 Proteins in action: Oxygen transport and storage by haemoglobin and myoglobin. Learning Objectives At the end of your study relating to this topic you should be able to: Recognise and describe the structures of myoglobin and haemoglobin. Explain oxygen binding to globins and how it is measured. Contrast haemoglobin with myoglobin, referring to both structure and function. Describe cooperativity in haemoglobin, including conformational change. Explain the relationship of allostery to cooperativity and how both underlie control of protein activity. Explain examples of physiologically relevant changes in haemoglobin function under specific conditions. Textbook references: Chapter 4, “The structure of proteins” pages 69-75 Biochemistry and Molecular Biology, 6th edition by D. Papachristodoulou, A Snape, W.H. Elliot and D.C. Elliot 48 Haemoglobin (Hb) Haemoglobin is the predominant macromolecule inside red blood cells, and gives blood its characteristic red colour. The haemoglobin molecule consists of two distinct parts: a protein known as globin and a non-protein unit called haem. At the centre of the haem is an atom of iron in the ferrous (Fe2+) state. This iron atom is central to the reversible carriage of oxygen around the body. Haemoglobin exists in two general conformations. When oxygen is being carried, haemoglobin is in the R-state, and referred to as oxyhaemoglobin. When haemoglobin is not carrying oxygen (i.e., on its return to the lungs), it is in the T-state, referred to as deoxyhaemoglobin. Figure 11-1. The structure of haemoglobin (Campbell, Farrell, Note: American English spelling is heme and McDougal, p.93) and hemoglobin Myoglobin (Mb) Myoglobin is biochemically related to haemoglobin. It is also a globin with a haem attached, but has a slightly different structure and thus a different function. Myoglobin does not circulate in blood, but is found inside muscle cells, where it provides a reservoir of oxygen to indirectly energise muscular contraction. Myoglobin is also red and this, rather than retained blood, gives isolated muscle (such as meat) its colour. The main difference in structure is that haemoglobin exists as a tetramer, i.e. four globins attached together in a quaternary association, whereas myoglobin molecules exist as monomers. As a result, myoglobin binds oxygen much more tightly than haemoglobin does. Figure 11-2. The structure of A) myoglobin b) haem (Voet, Voet & Prat, Figures 7.2). 49 Globin: the protein component of haemoglobin and myoglobin A recurring theme in protein biochemistry is that molecules are both ‘similar’ and ‘different’ depending on one’s point of view! Two different types of globin (called α and β) are found in haemoglobin, with another type in myoglobin. Note that the names α and β globin have nothing to do with α-helix or β-sheet. The different globins have different amino acid sequences, but are similar in having the same characteristic shape, with 8 regions labelled A-B-C-D-E-F-G-H that are α-helical, folded into a tertiary shape known as the globin fold. The oxygen binding properties of haemoglobin and myoglobin The main function of haemoglobin (Hb) is to transport oxygen from the lungs to the tissues. Haemo- globin contributes to other bodily activities, such as assisting in the transport of carbon dioxide from tissues to the lungs, but we will concentrate on the oxygen transport role. Figure 11-3. A comparison of the oxygen-binding behaviour of myoglobin and haemoglobin. The oxygen- binding curve of myoglobin is hyperbolic, whereas that of haemoglobin is sigmoidal. Myoglobin is 50% saturated with oxygen at 1 torr partial pressure; haemoglobin does not reach 50% saturation until the partial pressure of oxygen reaches 26 torr. Study the ‘oxygen saturation curve’ of haemoglobin (the curve is for pure haemoglobin in the laboratory and is a little different for whole blood). As oxygen is made available, very little happens at first. At 10 units of oxygen pressure haemoglobin has absorbed hardly any oxygen; even at 26 units pressure it is only half-saturated, and it reaches full saturation at 100 units. What is really important about this curve is the reverse: as blood flows through the lungs where the oxygen pressure is 100 units, it becomes saturated with oxygen, but in the tissues where the pressure may be as low as 20 units, the haemoglobin reverts to being 40% or 50% saturated, releasing much of its bound oxygen. Oxygen carriage is reversible. Note that the saturation curve is slightly S-shaped, or sigmoidal, which is (usually) associated with the molecular phenomenon of ‘co-operativity’. Myoglobin on the other hand becomes half-saturated with the mere trace of oxygen and is fully saturated at 10 units. Myoglobin could not function as a transporter of oxygen in the blood. It would become 100% saturated with oxygen in the lungs and remain almost 100% saturated at the tissues. Obviously in this scenario, almost no oxygen would be released. In highly active muscle however, it is useful for storing oxygen and then releasing it when supplies become desperately low. Myoglobin’s curve shows no sign of co-operativity. 50 Haem: where oxygen is carried Figure 11-2 (b) shows how haem comprises four rings, called pyrrole rings, linked together by –CH= (methine) bridges. The whole structure is called a porphyrin ring. Attached peripherally are methyl-, vinyl- and propionyl- side groups. There are 15 possible isomers, or ways of arranging these side groups, but the same arrangement is always present in human globins, known as protoporphyrin IX, because it is 9th on the list of isomers. The business end of haem, and of globins, is the ferrous iron (Fe2+) held between the four nitrogens. This iron carries a molecule of oxygen (O2) but does not become oxidised to Fe3+. Oxygen does not bind to ferric (Fe3+) haem. The haem Fe2+ is oxygenated rather than oxidised. We rationalise that the purpose of the haem-globin combination is to obstruct O2 from oxidising Fe2+, allowing only sufficient access as to be attached reversibly. Figure11-4. The structures of a) deoxyhaemoglobin and b) oxyhaemoglobin (Voet, Voet, & Pratt, figure 7-5). What causes co-operativity? Tetrameric haemoglobin displays co- operativity but monomeric myoglobin does not, because the key to co-operativity lies in the quaternary association of the four globins in haemoglobin. Normal haemoglobin in the human adult has two α and two β globins arranged in a symmetrical structure. The globins are not merely stuck to each other statically, they slide a little and interact such that the entire tetramer can exist in two states, known as R and T. The R state haemoglobin structure is such that it has a high affinity for oxygen while the T state haemoglobin configuration has low oxygen affinity. The important point is that switching between the R and T states triggers all four globins to Figure 11-5. Orientation between helix F, the porphyrin ring be in the same state, there is not a mixture. and haem iron in the R- and T-states (Voet and Voet, p333) 51 More importantly still, O2 binding triggers a switch to the R state, which happens to favour O2 binding, whereas O2 release triggers the switch to the T state that further assists O2 release. This happens because O2 binding to the iron causes a distortion in the haemoglobin structure that is transmitted from atom to atom, first through histidine F8 and then other amino acids and helices. This favours a change of state from T to R in one globin unit, which then induces a change in the remaining subunits. Thus, in the lungs O2 uptake favours more O2 uptake, and in the tissues the release of O2 favours the release of more O2 (i.e. the process is co-operative). As can be seen in figure 11-5, deoxyhaemoglobin has a dished haem, while in oxyhaemoglobin, oxygen flattens the haem, and pulls histidine F8 and helix F toward the binding site. Anything that keeps helix F away from the binding site will weaken oxygen binding. In red blood cells, the shift from R state to T state is assisted by the presence of 2,3-bisphosphoglycerate (BPG). This small molecule, which has 5 negative charges, binds to a positively charged site in the centre of the haemoglobin tetramer. The majority of the positive charge of the pocket is made up by three positively charged amino acids (His 2, His 143 and Lys 82, also see Figure 11-10) in either of the two β subunits (thus contributing a total of six positive charges). This positively charged pocket is geometrically much more compatible with BPG when haemoglobin is in the T state, thus BPG binds bind to T state haemoglobin almost exclusively. Figure 11-6. The binding of BPG Binding of BPG to T state haemoglobin stabilises the T state and is a to deoxyhaemoglobin (Campbell, major player in achieving Farrell and McDougal, p.97). the sigmoidal curve of haemoglobin oxygen binding. This means that once T state haemoglobin has formed and BPG bound to it, it is held in the low oxygen T state, not being able to revert back to the R state. Later in this paper you will see how BPG is produced when glucose is broken down in for energy production. (It is in fact 1,3- BPG that is formed but a proportion of it becomes 2,3-BPG). Think about when and where in your body you would need haemoglobin to bind oxygen with high or low affinity and how BPG fits into this! When the T state haemoglobin circulates back to the lungs, the high concentration and pressure of oxygen pushes the BPG off, switching the haemoglobin back to the R state. A molecule that binds at a site other than the active site or functional site of a protein and affects protein function is called an allosteric regulator. The site to which it binds is called an allosteric site (meaning another site). The sigmoidal curve of co-operativity is characteristic of an allosteric mechanism. BPG is an allosteric regulator of haemoglobin function (i.e. it binds somewhere other than the haemoglobin functional site (haem Fe) and affects protein function). Figure 11-7. The effect of BPG and CO2 on haemoglobin oxygen binding (Mathews, van Holde and Ahern, p.230). 52 Cooperativity is only prominent in the presence of allosteric inhibitors of binding, so, in this case, inhibitors of oxygen binding. Allosteric inhibitors BPG, CO2, and H+ all stabilise the T-state of haemoglobin. This helps to unmask cooperativity. This can be seen in figure 11-7, where the more allosteric inhibitors present, the higher the degree of cooperativity. Figure11-8. The effect of pH on haemoglobin oxygen binding (Bohr effect). 53 Special haemoglobins Haemoglobin containing two α and two β globins (α2β2) is characteristic of most adults, but embryonic, foetal and neonate haemoglobin subunit composition varies (Figure 11-9). For about the first 8 weeks of gestation, α is represented by a form with a slightly different sequence, known as ζ (zeta), and β is represented by ε (epsilon). Gradually α rises to full production but β is mostly represented by yet another form called γ (gamma), the full changeover to β happening around the time of birth. A trace of a β variant known as δ (delta) is also present in the adult. Whichever of these variants is utilised, the normal functional haemoglobin always comprises two α-like and two β-like variants. The rationale for the existence of foetal haemoglobins is that they have a slightly higher affinity for oxygen, thereby favouring the transfer of O2 from mother to foetus. Figure 11-9. The progression of human globin chain synthesis with embryonic and foetal development. Note that any red blood cell contains only one type each of α- and β-like subunits (Dickerson and Geis, p.85). 54 Figure 11-10. The binding of BPG to deoxyhaemoglobin. His and Lys are positively charged amino acids. In the γ subunit there is a uncharged Ser in place of His 143. (Figure 7.17 from Biochemistry, 7th ed). In the foetal γ subunit, the His 143 is a Ser (serine) amino acid, which makes the BPG pocket carry two less positive charges. Consequently BPG binds less well and the R state high oxygen affinity form of haemoglobin is favoured. Figure 11-11. Comparison of oxygen saturation curves for maternal and fetal haemoglobin. 55 Sickle cell and Haemoglobin S In sickle cell anemia, the abnormal haemoglobin (Haemoglobin S) sticks together when it is in its deoxygenated form. Haemoglobin S is cause by a point mutation of the 6th amino acid in the β chain. The polar positively charged glutamate, located on the surface of the protein, is replaced by the non- polar uncharged valine. This change makes Haemoglobin less soluble and in the deoxygenated form the valine binds to a hydrophobic pocked of another deoxygenated haemoglobin S protein, forming a long polymer of haemoglobin S proteins. The polymers essentially stretch the red blood cell out, giving it its sickle shape. β1 α1 β1 α1 β1 α1 β1 α1 β2 α2 β2 α2 β2 α2 β2 α2 Oxyhaemoglobin A Deoxyhaemoglobin A Oxyhaemoglobin S Deoxyhaemoglobin S α1 α2 α1 α2 α1 α2 β1 β2 β1 β2 β1 β2 β1 β2 β1 β2 β1 β2 α1 α2 α1 α2 α1 α2 Deoxyhaemoglobin S polymerises into filaments Figure 11-12 Different structural forms of haemoglobin A (normal adult haemoglobin) and haemoglobin S in their oxygenated and deoxygenated forms. Figure 11-13. Sickle cells block capillaries. (Easy Learning Genetics: Part II – Genetic Disorders and Genes involved.) 56 Lectures 13 & 14 Activation and inhibition of proteins. Lectures 13 and 14 will be given by the Department of Pharmacology and Toxicology. Learning Objectives At the end of your study relating to this topic you should be able to: Explain the steps that cause protein activation or inhibition. Compare enzymes and receptors. List the different receptor classes and compare their signal transduction mechanisms. Define the terms: receptor, ligand, agonist, antagonist. Outline the signal transduction mechanisms for the following specific ligand/receptor examples: Insulin receptor Glucagon receptor GLP-1 receptor Textbook references: There are no specific readings associated with these lectures. 57 Proteins as Drug Targets Many drugs currently in use work by targeting regulatory proteins. These include carrier molecules, enzymes, ion channels and receptor proteins. Receptors are normally found on the cell surface and typically span across the cell membrane (transmembrane proteins). There they act as recognition macromolecules allowing chemical detection and communication across the cell membrane. A chemical substance that binds to a receptor protein is called a ligand. In more generic terms a ligand is defined as a smaller molecule that binds to another larger molecule. A ligand that interacts with a receptor to produce a measurable biological response, i.e. activates the receptor, is known as agonists. In contrast, ligands that interact with a receptor to block a biological response are known as antagonists (equivalent to inhibitors of enzymes). Therefore, agonists are drugs that mimic endogenous messengers, whereas antagonists are drugs that block endogenous messengers. The common steps of receptor activation and inhibition To activate or inhibit a receptor, a ligand must bind the receptor protein. Ligands can be endogenous (produced in our body) or exogenous (not produced in the body but are introduced). Medicinal (or recreational) drugs and toxins are exogenous ligands. The first step in this process involves the ligand travelling from its source to the receptor. The ligand then binds to the receptor. This binding event is known as “reception.” Most ligands do not enter the cell. This is because most receptors are located on the outer cell membrane, and therefore act as sensors of the local extracellular environment. As a result of ligand-binding, there receptor protein undergoes conformation changes. Following reception, the structural changes of the receptor protein to which the ligand bound will become activated or inhibited, depending on whether the ligand was an agonist or antagonist. Regardless of which, there will be a change in cellular response. The nature of this cellular response is dependent on the receptor that has been activated or inhibited. Figure 13.1. Overview of the signal transduction process. Following activation, a chain of intracellular events will be initiated. This is called signal transduction; a chain of events where messages (in the form of various molecules) are passed on through the cell, ultimately leading to a cellular response (figure 13.1). Inhibition of a receptor prevents the signal transduction process from taking place, thereby inhibiting the cellular response. Enzymes vs. Receptors – What’s the difference? Enzymes and receptors share many similarities. They both bind something, and can both be membrane bound or free in the cytosol, they both show specificity (that is, the active site/ligand binding site and substrate/ligand are complementary to one another). Both enzymes and receptors can be activated and inhibited and are extensively used as medicinal drug targets. So, what are then are the differences? An enzyme generally has a single active site where one substrate molecule binds. This substrate molecule is then changed into a product. In contrast, receptors may have one or multiple ligand binding sites. The ligand binds to activate or inhibit a cellular response, the ligand is then released unchanged. 58 Receptor Classes Just like enzymes, not all receptors are the same. There are four types of physiological receptors. In these two lectures we will discuss the three most common types (figure 13.2). Membrane proteins coupled to ion channels (known as ligand-gated ion channels) Membrane proteins coupled to G proteins (known as G-protein coupled receptors, or GPCRs for short) Membrane proteins coupled to enzymes that phosphorylate, i.e. kinases (e.g., receptor tyrosine kinase, RTK) Receptors that regulate gene expression (known as nuclear receptors) Although the structures and processes of these receptors are different, they all share the same general steps of activation/inhibition described above. The details change depending on the receptor type, but the basic outline is the same. Figure 13.2. The three major classes of receptors. Figure 13.3 outlines the overall process of activation of the major receptor classes. You should be familiar with the overall process of ligand-gated ion channels, GPCRs, and kin ase-linked receptors. You do not need to know any details regarding nuclear receptors. Figure 13.3. Overview of the signal transduction process of the four major receptor classes. 59 Signal Transduction overview Following binding of a ligand to a receptor, a signal is passed from the membrane into the cell, eliciting a cellular response. Exactly how that signal is delivered to the cell depends on the type of receptor that has been activated. For a ligand-gated ion channel, where the receptor protein in forming a channel across the membrane that can be opened and closed, ligand binding opens the channel, causing ions to flow into the cell. As ions are charged molecules, the charge difference between the inside and outside of the cell changes. This in turn, leads to an immediate cellular response (by creating and action potential). Due to this, the timescale by which an ion channel generating a response is in milliseconds. This type of receptor is found extensively within the brain, where it is used to transmit signals during neurotransmission. A G-protein couple receptor (GPCR) involves a much more complex signal transduction pathway. When an agonist ligand binds to the GPCR, the resulting conformation changes of the transmembrane protein are transferred to a G protein located on the intracellular side of the cell membrane. (It is called a G protein because it also uses the energy molecule GTP to become activated). As a result of this conformational change, one of its subunits, stimulatory G protein (Gas), binds to a membrane-bound enzyme called andenyl cyclase (ak.a. andenylyl cyclase), activating it. Activated adenyl cyclase then synthesises a second messenger molecule (often cAMP, made from ATP). Second messengers are intracellular signalling molecules released or synthesised by the cell in response to exposure to extracellular signalling molecules—the first messengers. Second messengers trigger physiological changes at cellular level. They can trigger the intracellular response as they are not bound to the membrane and are therefore free to move through the cell. The second messenger will activate the next relay molecule in the pathway, which activates the next relay molecule, and so on until the cellular response is achieved. This signalling pathway commonly involves activation of the next relay molecule by phosphorylating it; the relay molecules are kinases (enzymes that phosphorylates). It only takes seconds for the signal to be relayed through the pathway to generate the cellular response, which commonly is turning a protein ‘ON’ or ‘OFF’ by phosphorylating it. Receptor tyrosine kinases (RTKs) use phosphorylation of an adaptor protein to achieve their goal. Upon ligand binding, the RTK phosphorylates an adaptor protein that is bound to the membrane on the intracellular side in a similar way that the G protein is for GPCRs. This begins the signa