Proteins Amino Acids 1 Lecture.pdf

Contact Information: Dr. Ed Randell PhD DCC FCACB FAACC Clinical Chief and Professor, Lab. Medicine, Eastern Health Authority & Faculty of Medicine, Memorial University Rm 1J442 Laboratory Medicine Eastern Health St. John's NL Office: 709-777-2164 Cell: 709-725-0616 Fax: 709-777-2442 Linked lecture...

Contact Information: Dr. Ed Randell PhD DCC FCACB FAACC Clinical Chief and Professor, Lab. Medicine, Eastern Health Authority & Faculty of Medicine, Memorial University Rm 1J442 Laboratory Medicine Eastern Health St. John's NL Office: 709-777-2164 Cell: 709-725-0616 Fax: 709-777-2442 Linked lecture objectives appear on relevant slides. These have information essential to learning objectives (for review purposes). Reference and Background: Rodwell, V. W., Bender, D. A., Botham, K. M., Kennelly, P. J., & Weil, P. A. (2018). Harper's illustrated biochemistry. New York (NY): McGraw-Hill Education. Chapters 2 & 3 (Electronic Access available from MUN Library) ER’s slide notes are provided to assist with independent study by learners. Review of these are not essential to meeting objectives but may assist with understanding slide content by providing additional context. 1 2 Two facts are of fundamental importance to building your understanding of biochemistry (the chemistry of life): 1- Life is maintained by chemical processes; and 2- Life exists as molecules interact and react in water. All life as we know it requires the use of biochemicals to construct, tissues, and organs; and the use of biochemicals in highly controlled chemical reactions. Understanding life at the molecular level requires a knowledge of the types of biochemical molecules involved and the types of reactions they participate in. A good understanding of medical biochemistry is essential to understanding the fundamental basis for biological health and disease. From a biochemistry perspective health exists when all intra- and extracellular biochemical reactions occur at rates that support maximal probability for physiological survival. Disease is the product of abnormalities in biochemical processes, reactions, and molecules. 3 Proteins are linear sequences of α-amino acids bound together by peptide bonds. The proteins are involved in transport, protection and defense, storage, control and regulation, catalytic function (as enzymes) and contractile movement, and in structural functions. 4 Amino acids are the building blocks of proteins. In most amino acids the α-carbon atom is a chiral carbon. A molecule is chiral if there is no internal plane of symmetry. A carbon atom is chiral if it has four different non-identical groups attached to it. Note this in the illustration. α-amino acids have attached to the α- carbon a(n): 1-Carboxyl group. 2-Hydrogen atom. 3-Amino group. 4-Side chain (R-group) are different from one amino acid to the next. It is part of what distinguishes one amino acid from another. The amino acid Glycine is the exception (no chiral carbon). It has a second hydrogen atom as a side chain and does not meet the requirements for a chiral alpha carbon. 5 In the case where a chiral carbon is present the image and its mirror image are not superimposable 2 different optical isomers (relative to the α-carbon) arise from most amino acids: levorotatory; L; - dextrorotatory; D; + In other words, there are two forms to almost every amino acid. But only L isomers are used during protein synthesis. Many different D-amino acids occur in nature but are not used in protein synthesis (important in bacterial cell walls). 6 The Peptide Bond The peptide bond is a covalent bond formed by reaction of α-amino group of one amino acid with α-carboxyl of another amino acid. Water is eliminated in the process. Amino acid residues (side chains plus α-carbon) remain intact after the peptide bond is formed. In a peptide or protein the peptide bond links consecutive amino acids along its primary sequence. NOTE: how the peptide backbone forms a continuous chain. 7 Amino acids, peptides and proteins are three dimensional structures. The peptide bond is strong, rigid, and significantly restricts rotation (it has a partial double bond character) and, hence, restricts peptide and protein structure because it behaves more like a double bond than a single bond. The restricted rotation is cause by resonance stabilization because of sharing of electrons along the carbonyl oxygen and amino nitrogen. Once a peptide bond forms the C1-CO-NH- αC2 all occur within the same plane – this affects the three-dimensional structure of peptides and proteins. Also, in this structure α-carboxyl and α-amino groups are not available for chemical reaction but can participate in hydrogen bonding with other groups that are closely located following protein/peptide folding. 8 The size and shape of side chains (R-groups) affect the degree of rotation and large R-groups cause steric interference. This causes the α-carbons and side chains of consecutive amino acids to more commonly orient in trans conformation in which the R groups are oriented far away from each other to maximize distance between them and to minimize the steric interference caused by adjacent amino acid side chains (R-groups). The cis conformation is unstable because of steric interference and occur less frequently in peptides and proteins. 9 Aspartame is made from a dipeptide (two amino acids) and a methyl group that comes from methanol (CH3-OH). 10 Oxytocin is a 9 amino acid peptide hormone that stimulates uterine contractions during childbirth; stimulates lactation, but also affects behavior. It is produced in various social interactions and behaviors including, bonding between parent and infant, bonding/attraction between partners, and in sexual arousal. It contains a disulfide bridge in its structure. The free sulfhydryl of cysteine readily forms disulfide bridges as in the example above. And the various arrows point to the peptide bonds that tie together this 9 amino acid peptide. You will note that the peptide backbone is a repeat of three atoms (amino N – carbonyl C - αC). 11 12 Water is essential for life and is the media in which life exists. The properties of molecules in water are major drivers for biochemical reactions that sustain life. These properties include: 1) the polarity of water (and how this impacts molecules interacting with it); and 2) acid-base properties. Water is Polar – Polar molecules are relatively positively charged on one side and negatively charged on the other. Note that the water structure is bent at about 105 o. The polarity of water allows it to: 1- Form Hydrogen Bonds – Between hydrogen, typically covalently bound to an electronegative atom like oxygen or nitrogen, and another electronegative atom on the same or different molecule. Hydrogen bonds are weak bonds of about less than 1/10 the strength of covalent bonds. 2- Participate in four hydrogen bonds: Its geometric arrangement forms a tetrahedral where bonds are constantly breaking and forming giving water its fluid character. 3- Stabilize and dissolve ions (charged atoms and molecules) 13 The polarity of water causes it to interact differently with different molecules. Note the interactions of water with chloride and with sodium. The negative chloride ion tends to interact with the slightly positive character of hydrogen to dissolve chloride. The positive sodium ion tends to interact with the slightly negative character of oxygen to dissolve sodium: Hydrophilic Interactions (shown)- “Water loving” molecules tend to dissolve in water and are polar. Fully charged ions and partially charged (polar) molecules can interact and dissolve in water. Molecules with electronegative ions are hydrophilic, polar, and include alcohols, amines, and carboxylic acids. Hydrophobic Interactions (not shown) - “Water fearing” molecules tend not to dissolve in water and are non-polar. Weak interactions occur with nonpolar molecules, but it is less thermodynamically stable for water molecules to interact with nonpolar molecules than with other water molecules. This leads to hydrophobic compounds (or hydrophobic parts of compounds) to self-associate. Ionic Interactions (not shown) – Attractive forces between opposite charges and repulsive between like charges. 14 Amino Acids are classified by Polarity, Hydrophobicity, and Hydrophilicity. Polar compounds participate in hydrogen and ionic bonds. Hydrophilic (water loving) molecules form hydrogen bonds with water. Polar amino acids are hydrophilic and include those with ionizable side chains, or side chains that form hydrogen bonds. Hydrophilic amino acid side chains are frequently found extending out from a protein’s surface and interacting with the aqueous solution. 15 Hydrophobic (water fearing) molecules are repelled by water. Amino acids with non-polar side chains participate in hydrophobic interactions. Hydrophobic amino acid side chains gather to protein interiors to escape aqueous environments or project outward into the hydrophobic lipid membranes. Think about trying to mix oil (hydrophobic and non-polar) and water (polar). 16 Water has both acid and base characteristics. Acids are proton (hydrogen ion) donors and bases are proton acceptors. The composition of water in its normal form involves partial dissociation into its acidic and basic counterparts to reach an equilibrium. The concentration of each at equilibrium is indicated in the figure. For ease of understanding, we simplify these very small numbers indicating hydrogen ion concentration by negative logistic mathematic transformation and expressing it as a “negative log10 term” and add a “p” to indicate this. In other words, we speak about the degree of dissociation of water in terms of pH and, as indicated in the figure, water dissociated to give a hydrogen ion concentration equivalent to 10-7 or pH 7 and is said to be neutral because the positively charged hydrogen ion concentration is equal to the amount of negatively charged hydroxide ion concentration. As the concentration of dissolved hydrogen ion increases and decreases it affects the properties of water and compounds, like amino acids, peptides, and protein dissolved in it. pH provides information about hydrogen ions ([H+]) in aqueous solution. In summary, pH indicates the - log10[H+]. 17 17 Strong acids when dissolved in water dissociates completely to give up all its hydrogen ions. A strong base dissociates completely of its hydroxide ions. Hydrochloric acid is an example of a strong acid, and sodium hydroxide is an example of a strong base. Weak acids do not dissociate completely. A weak acid (HA) consists of a conjugate base (A-) and H+ (hydrogen ion) that are in equilibrium in aqueous solution. They increase H+ and reduce pH (make solution more acidic) in water. Carboxylate groups are weak acids – they tend to release protons better than water. A weak base, in contrast, has the effect of binding free H+ to it and thereby pulling the reaction in favor of more HA. A weak base will decrease H+ and raise pH (make solution more basic or alkaline) Amino groups are often weak bases – they tend to bind to protons better than water and by doing so tend to carry a positive charge. Amino acids have at least one carboxylate group and one amino group. Meaning that part can release a proton (hydrogen ion) while the other part tends to mop up protons. 18 18 So how does pH apply to biological fluids? Notice the low pH of gastric juices - the presence of high concentrations of hydrochloric acid is responsible for this low pH. What do you think is responsible for the pH of other fluids. Is it caused by strong acids and bases or weak acids and bases? Which chemical groups do you think are involved imparting the acidic and basic characteristics? Answer: For most fluids, the acids and bases are weak acids and bases. Carboxylate and amino groups are major contributors to the acidic and basic characteristics of the various fluids. You will note in the next section that carboxylate and amino groups are integral to the structure of amino acids and these groups can affect the local environment by acid and base interactions. 19 19 A weak acid (HA) has a conjugate base (A-) and H+ (Hydrogen ion) in equilibrium in aqueous solution. Ka is the acid dissociation constant. It is given by an equation where HA (acid) is in equilibrium with H+ + A- (conjugate base), or Ka=([H+][A-])/([HA]). pKa indicates the -log10Ka. pKa: Determines the pH at half equivalence point where the concentration of weak acid and its conjugate base are equal. The equivalence point is the point at which all acid is titrated or neutralized. When [HA] = [A-], pH = pKa and [H+] = Ka. Acid strength is determined by the amount of H+ released when an acid dissolves in water. Stronger acids are more dissolved and have larger Ka. pKa is a more convenient measure of acid strength than Ka. The lower the pKa, the stronger the acid. pKa depends on the local environment. Nearby polar and ionic groups can impact the actual pKa making it higher or lower depending on local conditions. The relationship between a weak acid and its conjugate base is described by the Henderson– Hasselbalch equation (above). It is a mathematical transformation of the relationship between a weak acid, conjugate base, and hydrogen ion dissolved in water, and the resulting pKa. The equation is useful for predicting properties of buffer solutions that control pH allowing biochemical reactions to occur at appropriate rates. 20 20 When pH of a solution is less than the pKa of a weak acid, the protonated form of the weak acid is present at higher concentration. When the pH of a solution is greater than the pKa of a weak acid, the deprotonated form is present at higher concentration. Control of pH is essential to maintaining amino acid, peptide, and protein structure, to biochemical reactions and hence survival. Going outside the narrow range at which pH must be controlled can be detrimental to biochemical processes, structures, reactions and life. Buffers control pH. Buffers are therefore critical for life. Buffering of blood is mainly by hemoglobin (a protein) and bicarbonate. But both work essentially the same way to maintain pH. By definition a buffer is a solution that resists changes in pH when small amounts of a strong acid or strong base are added to it. Biochemical reactions produce acids and bases. A buffer consists of the combination of a weak acid and its conjugate base dissolved in solution. The effectiveness of a particular buffer depends on the pH of the solution and the relative concentrations of weak acid and conjugate base to each other. Buffering occurs best at a pH near the pKa of a weak acid and is greatest within 1 pH units of pKa. At pKa the concentration of weak acid is equal to the concentration of weak base. 21 The side chain (R-group) defines the properties of the amino acid and distinguishes one amino acid from another. At physiological pH, carboxyl group loses its H+ and amino group gains a H+ to produce both a -ve and a +ve charge on the same molecule. The amino acid α-carboxyl group is a weak acid (H+ donor). The amino acid α-amino group is a weak base (H+ acceptor). Amino acid R-groups can be either weakly acidic, basic or neutral. This slide illustrates the relationship between added base and pH for the amino acid alanine in an acid-base titration curve. Notice that both the α-COOH and α-NH2 groups are capable of ionizing (taking on a charge). Free amino acids have at least 2 weakly ionizable groups. Based on the pKa, the carbonyl group is a stronger acid than the amino group and will be deprotonated (or negatively charged) at physiological pH. The α-amino group would be protonated at physiological pH but the net charge of the entire molecule is zero (in the example provided). The net charge on amino acids in solution or in peptides and proteins depends on the pH of the solution. The pH at which the sum of all charges of an amino acid or protein is equal to zero is called its isoelectric point (or pI). When there are two ionizable groups the pI is equal to the average of the 2 pKas (that is, pK1 and pK2 in the figure above). 22 Now we will cover the classification of amino acids based on their chemical properties. Amino acids are distinguished by their side chain structure and characteristics. We will discuss the 20 main proteogenic amino acids and briefly indicate which ones have side chains that have predominantly polar, non-polar properties, or are weak acids and weak bases; and identify other distinguishing structural properties. We will also do this by grouping amino acids by structural characteristics. R-groups determine the specific properties of amino acids. 23 Note that amino acids are written in abbreviated form using a three-letter or one-letter notation. It is not essential that you remember these, but the three-letter abbreviation aligns well with the long form name of the amino acid. The first group we will cover are aliphatic amino acids. These amino acids have side chains that do not bind or release protons, nor do they participate in hydrogen or ionic bonds. It is reasonable to expect these side chains to favor positioning in the interior of proteins (favoring hydrophobic interaction). Alanine: Important for glucose metabolism; helps maintain nitrogen balance via the alanine cycle. Glycine: Because of the small size of the glycine side chain (H) it is often found at sharp bending points in the amino acid sequence in proteins facilitating flexibility. Glycine is also an inhibitory neurotransmitter in spinal cord; involved in synthesis of other amino acids, nucleic acids (RNA, DNA) and bile; and necessary for collagen formation. Valine, Leucine, and Isoleucine are collectively called the branched chain amino acids (Remember this subgrouping it will come up again). All three of these are degraded by the liver and preferentially used by the muscle for energy. Neither of these three can be made in humans, making them essential in the diet. 24 The ring structure of proline gives it some special properties during the formation of secondary structure in proteins. Proline is the only amino acid out of the 20 that is formed as a secondary amine by covalent linkage of its alpha amino group with its side chain – making it an imine rather than an amine. We place it here as it shares similar non-polar properties to the previous five. Proline also gets hydroxylated in collagen with the aid of ascorbic acid (vitamin C) and promotes connective tissue health for bones, joints, tendons, and skin. The bending in the helical structure of collagen is assisted by proline. The three chains can become closely packed because of glycine and the small size of its side chain. Note that the rigid structure of proline makes it incompatible for the alpha helix (later). The collagen helix is a special case. 25 And now another group of amino acids: The hydroxyl groups of serine, threonine, and tyrosine (see later – tyrosine has an aromatic not aliphatic side chain) can serve as an attachment site for phosphate and other groups in post-translational modifications (to be defined in next lecture). These side chains are polar and participate in hydrogen bonding. The hydroxyl groups also serve as good nucleophiles or reactive centers in the active sites of enzymes. The attachment of phosphate groups to amino acid side chain hydroxyl groups can activate/inactivate enzyme activity and serves important functions in regulating enzymes inside the cell. The hydroxy group of Serine and Threonine can also serve as attachment sites for oligosaccharide chains forming glycoproteins (visited again next lecture). The amino acids asparagine and glutamine are related to aspartate and glutamate but have an amide group at the side chain carbonyl. (Aspartate and glutamate will be covered shortly) Asparagine can also become an attachment site for oligosaccharide chains in glycoproteins. Glutamine is present at relatively high levels compared to other amino acids as it serves to detoxify ammonia by carrying it relatively benign form in its structure. (discussed in later lectures) 26 Four of the amino acids have aromatic side chains. Two are on this slide, phenylalanine and tryptophan. Note the aromatic ring structure on these two amino acids. Both side chains are non-polar. The hydroxyl group on tyrosine is an attachment site for other groups such as phosphate. The hydroxyl group makes tyrosine side chain polar. Tyrosine: Is a precursor to epinephrine, norepinephrine and dopamine and is necessary for the production of thyroid hormones, and melanin for skin, hair, and eye pigmentation. We will discuss histidine (another aromatic amino acid) with the basic side chain amino acids. 27 Two amino acids contain a sulfur atom in their structure. Methionine has a much more hydrophobic side chain because of the methyl group cap and is an important source of methyl groups (as in S-adenosyl methionine) for many biosynthetic reactions (neurotransmitters, creatine, carnitine, phospholipids, etc.). Cysteine is more polar and has a reactive sulfhydryl group. This reactivity is important at the active site of enzymes and for formation of covalent disulfide cross-links in proteins. Disulfide bridges occur by oxidation of the sulfhydryl groups of two cysteine side chains. Much of cysteine is found in blood as cystine (two cysteines bound through a disulfide bridge) 28 As an example, note the disulfide bridge in Oxytocin. There are many other examples as this occurs frequently. 29 29 The side chains of glutamate and aspartate are proton donors. These are also called acidic amino acids. Other amino acids can also donate protons but only in very special conditions and not at physiological pH. Aspartic Acid and glutamic acid side chains are involved in acid/base catalysis at enzyme active sites. This next group are called basic amino acids. The imidazole ring of histidine and the guanido group of arginine both exists as resonance hybrids with the positive charge distributed among the nitrogens of the side chain. Histidine side chain can serve as a buffer at physiological pH and has an isoelectric point of about 7.6. The imidazole group of histidine allows it to act as either a proton donor or acceptor at physiological pH serving in either acid or base catalysis. This property permits the use of histidine at active sites in enzymes. This is important for the histidine side chain to function in O2 and CO2 exchange and H+ binding in hemoglobin. At physiological pH lysine and arginine are fully protonated, ionized, and positively charged. 30 There are many other amino acids not occurring in protein but play important physiological functions. Here we show a couple in the example: Taurine and Homocysteine. Taurine is conjugated with bile acids in the liver (Read your Red Bull and other energy drink label – you will find it often listed) and used to assist fat digestion. Homocysteine serves as an intermediate during biosynthesis of cysteine or for regeneration of methionine. Homocysteine is also a biomarker for deficiency of certain B-vitamins (discussed in later lectures). In addition to the 20 proteogenic amino acids, another exception worthy of mention is Selenocysteine. The amino acid is placed in proteins under special circumstances, but we will not be discussing it further here. 31 Cystinuria - StatPearls - NCBI Bookshelf (nih.gov) 32 33 ) 34 Cystinuria: An Overview of Diagnosis and Medical Management - PMC (nih.gov) 35 This will be filled at time of class. 36