BCH 208: Enzymes and Biocatalysis PDF

BCH 208: Enzymes and Biocatalysis  Introduction Enzymes are protein molecules that are present in all living things. They speed up and target chemical reactions, in many cases increasing the rate of reaction millions of times. For example, they aid digestion, met...

BCH 208: Enzymes and Biocatalysis  Introduction Enzymes are protein molecules that are present in all living things. They speed up and target chemical reactions, in many cases increasing the rate of reaction millions of times. For example, they aid digestion, metabolise and eliminate waste in humans and animals, and play a crucial role in muscle contraction. Enzymes have been used unknowingly in food production, e.g. dough making, for centuries. They can be obtained by extraction from plants or animals or by fermentation from micro-organisms. Enzymes are the body’s labor force to perform every single function required for our daily activities and are required to keep us alive  Introduction Life depends on a well-orchestrated series of chemical reactions. Many of these reactions, however, proceed too slowly on their own to sustain life. Hence nature has designed catalysts, which we now refer to as enzymes, to greatly accelerate the rates of these chemical reactions. Today enzymes continue to play key roles in many manufacturing processes and are ingredients in numerous consumer products. Enzymes are also fundamental interest in the health sciences, since many disease processes can be linked to the aberrant activities of one or a few enzymes. Hence, much of modern pharmaceutical research is based on the search for potent and specific inhibitors of these enzymes as therapeutic targets. Introduction  Introduction From DNA to Protein through transcription and translation. The genetic code is universal such that a gene from one organism can be transcribed and translated in another organism. DNA holds all of the genetic information necessary to build a cell’s proteins. The nucleotide sequence of a gene is ultimately translated into amino acid sequence of the gene’s corresponding protein. 6  Introduction The DNA encoded sequence of amino acids gives four different structure of proteins: –Primary structure –Secondary structure –Tertiary structure –Quaternary Enzymes are proteins, they are at the quaternary level of protein structure This is obtained through folding 7 Introduction 8 Structure of trypsin enzyme Voet Biochemistry 3e © 2004 John Wiley & Sons, Inc.  Active site- – The region of enzyme that binds with the substrate and where catalysis occurs – All enzymes have one or more active sites Specificity- – Enzymes bind to their specific substrates in the active site to convert them to product(s) Regulation- – Enzymes can be activated or inhibited so that the rate of product formation responds to the need of the cell  Introduction Ordinary chemical reactions convert reactants to products but enzyme catalyzed reactions convert a SUBSTRATE or SUBSTRATES to products. Substrate is the substance that enzymes act on, and convert it into products. Some enzymes require no chemical groups for activity other than their amino acid residues in their active site. – Such enzymes are referred to as simple enzymes because they consists only of protein in their active form. Usually only a few of the 20 amino acid residues or side chains participate directly in enzyme catalysis. The best examples of polar amino acids participants in catalysis 1 are Cys, His, Ser, Asp, Glu and Lys. 1  Introduction Some enzymes may require an additional chemical compound called cofactor for proper function. – such enzymes are active only when they combine with their cofactors. A cofactor is a small non-protein molecules that is bound (either tightly or loosely) to an enzyme and is required for catalysis. Cofactors are either – inorganic ions or – small organic molecules Therefore we can also define a cofactor as an inorganic ion or a small organic molecule that aid in enzyme catalysis 12  Introduction The common metal ions cofactors required for complete enzyme activity include Mg2+, Cu2+, Fe2+, Fe3+, Ca2+, Zn2+, Mn2+, Ni2+ etc. Metal ions in the active site are attached to one or more amino acid side-chains. The metal ions have various functions, such as electron exchange and substrate stabilization.  Introduction The metal ions that are tightly bound to the enzyme molecule lead to the formation of metalloenzymes. – That is metalloenzymes contain firmly bound metal ions in their active sites (examples: Fe, Zn, Cu, Co). – The cations in metalloenzymes participate directly in catalysis Small organic molecules that serve as cofactors in enzymatic reactions are called Coenzymes. – Coenzymes are either loosely or tightly bound to the enzyme molecule. Coenzymes prepare the active site for proper substrate binding and/or participate in catalysis. They are required in small quantities and are not destroyed during the reaction. A coenzyme or a metal ion that is covalently bound to the enzyme protein is called a prosthetic group 12  The protein component of an enzyme without its necessary prosthetic group is called an APOENZYME or apoprotein. Whereas the active enzyme with its needed prosthetic group bound or covalently attached is called the HOLOENZYME – i.e. a completel catalytically active enzyme together with its cofactor. Holoenzyme = Apoenzyme + Cofactor (active) (inactive) (inactive) The functional unit of an enzyme is referred to as a holoenzyme. It is often made up of an apoenzyme (the protein part) and a coenzyme (the non-protein part). A zymogen or proenzyme is the precursor form of the enzyme in an inactive form An activator is any substance that increases the rate of an enzyme-catalyzed reaction. 15 16  Coenzymes use in enzyme catalyzed reactions are metabolically active forms of vitamins. The word vitamin comes from the Greek word “VITAMINE” which means vital for life –vita = life –amine = containing nitrogen (the first vitamins discovered contained nitrogen) Vitamins are organic compounds or molecules required in the diet in very small quantities for metabolism, normal growth and maintenance of health but cannot be synthesized in the body either at all or in 17  VITAMIN Although the body is able to produce part or even all of its requirements for some of the vitamins examples: – vitamin D from cholesterol and – niacin from tryptophan. However, vitamins must be provided in the diet. Classification of vitamins  The are usually converted to the metabolically active form called coenzyme which participate in biochemical reactions i.e. – many coenzymes are derived from water-soluble vitamins All of the water-soluble vitamins and two of the fat-soluble vitamins, A and K, function as cofactors or coenzymes. Coenzymes participate in numerous biochemical reactions involving energy release or catabolism, as well as the accompanying anabolic reactions.  Thiamin Thiamin is also known as vitamin B1. It was the first B vitamin to be identified It is a derivative of substituted pyrimidine and a thiazole linked by a methylene bridge. Thiamin is rapidly converted to its active form, thiamin pyrophosphate Thiamin pyrophosphate (TPP): coenzyme form  Biochemical functions of B1 In form of TPP, vitamin B1 is involved with the energy releasing reactions of carbohydrate metabolism Oxidative Decarboxyation Reactions  RIBOFLAVIN The name "riboflavin" comes from – Ribose (the sugar whose reduced form, ribitol,forms part of its structure) and – Flavin (the ring-moiety which imparts the yellow colour to the oxidized molecule) Riboflavin is also known as vitamin B2 It is a heterocyclic isoalloxazine ring attached to a sugar alcohol, ribitol.  RIBOFLAVIN The coenzymes forms of riboflavin are: – flavin mononucleotide (FMN) – Flavin adenine dinucleotide (FAD) Both coenzymes are formed in the intestine and liver The reduced forms of FMN and FAD are – FMNH2 and – FADH2, respectively. The enzymes that require FMN or FAD as cofactors are termed flavoproteins. Flavoproteins are involved in a wide range of redox reactions, e.g. succinate dehydrogenase and xanthine oxidase. CONVERSION TO THE COENZYME FORM FMN → ATP-dependent phosphorylation of riboflavin FAD → further reaction with ATP in which its AMP moiety is transferred to FMN.  FMN, FAD Coenzyme： Flavin mononucleotide FMN Flavin adenine dinucleotide FAD  Biochemical functions of riboflavin FMN and FAD serve as prosthetic groups of oxidoreductase enzymes Oxidative degradation of fatty acids (FAD is the prosthetic group of acylCoA the prosthetic group of acylCoA dehydrogenase. Oxidative deaminationof α-amino acids: 1.FMN: Prosthetic group of L-a.a.oxidase. 2.FAD: Prosthetic group of D-a.a. oxidase Riboflavin is also needed to help the body convert vitamin B6 and folate into active forms  Niacin Niacin consist of two vitamers: – nicotinic acid – nicotinamide It is also known as vitamin B3 It is unique among the B vitamins in that it can be synthesized from Tryptophan in the body. – So it is not strictly considered a vitamin. However, the conversion is inefficient and most people required dietary sources of both tryptophan and niacin.  Niacin Both nicotinic acid and nicotinamide can serve as the dietary source of vitamin B3. Niacin is required for the synthesis of the active forms of vitamin B3 – Nicotinamide adenine dinucleotide (NAD+) – Nicotinamide adenine dinucleotide phosphate (NADP+). Both NAD+ and NADP+ function as cofactors for numerous dehydrogenases In the structure of coenzymes, nitrogen atom of nicotinamide carries a positive charge due to formation of an extra bond Hence coenzymes are NAD+ & NADP+  BIOCHEMICAL FUNCTIONS The coenzymes: –NAD+ (NADH) and –NADP+ (NADPH) are involve in oxidation-reduction in Carbohydrate, Lipid protein metabolism  Carbohydrate Metabolism 1. Glyceraldehyde 3P-Dehydrogenase It catalyses the conversion of Glyceraldehyde 3P to 1,3 Bisphosphoglycerate  2. Lactate Dehydrogenase It catalyses the interconversion of lactate to pyruvate It occurs in anaerobic conditions (pyruvate to lactate in muscle and erythrocytes) Gluconeogenesis (lactate to pyruvate in the liver) 3. Pyruvate Dehydrogenase Complex Pyruvate dehydrogenase catalyses the conversion of pyruvate to acetyl CoA It utilizes NAD which is reduced to form NADH + H+ 87  Nature and Properties of Enzymes All enzymes are proteins or protein in nature (except for a small group of catalytic RNAs) – RNA molecules with enzymatic activity are called ribozymes – But not all proteins are enzymes They are present at extremely low intercellular concentration (e.g. 10-7 molar) Small quantities are needed to catalyze reactions They are not altered or consumed during reaction They are reusable. They are not part of the reactants or products. 35 Nature and Properties of Enzymes Cont… So any chemical reaction which proceeds in the presence of an enzyme will also proceed in the absence of the enzyme but at a much slower rate. – Enzyme accelerate reaction rates several fold compared to the rate of the same reaction in the absence of the enzyme. – Enzymes are highly specific – Interact with only one or a few of the substrates – They catalyze only one type of reaction 36  Chemical catalysts versus enzymes Regardless of their chemical nature, all catalysts including enzymes, exhibit several common features: Both increase the rate of chemical reaction Both lower the activation energy needed to start a reaction. Both are not consumed or used up during the reaction i.e. they remain unchanged. Both are needed in small or minute quantities. Both do not alter equilibrium of the reactions they catalyzed – They simply decrease the amount of time required to achieve equilibrium. – They have no effect on the position of the equilibrium and therefore do not change the amount of free energy released or the direction in which the reaction will proceed. Both act by forming transient complexes with substrate molecules, ordering them in a manner that facilitates their interaction. Both are reusable 37  Unique properties of enzymes Enzymes exhibit several unique features that distinguish them from chemical catalysts routinely encountered in inorganic chemistry. These unique properties of enzymes relate to their: They have active site Denaturation 93  Unique properties of enzymes cont… Higher reaction rates – The rates of enzyme catalysed reactions are typically several fold greater than those of the corresponding uncatalyzed reactions and several orders of magnitude than those of the corresponding chemically catalysed reactions. Milder reaction conditions – Enzyme catalysed reactions occurs under milder conditions such as; temperature, atmospheric pressure and near neutral pH. While chemical catalysts often require elevated temperature and pressure. Greater reaction specificity – Enzymes have greater degree of specificity with respect to the kind of reaction they catalyze as well as the identity of substrates acted upon than chemical catalysts. They have capacity for regulation – Ability to activate or inhibit enzymes can control which reactions occur and when. – The catalytic activities of many enzyme vary in response to regulations by substances other than their substrates and products. 39  Active site The active site of an enzyme is the region on the surface of an enzyme that binds the substrate and convert it into products. Active site of an enzyme possesses a unique geometric shape and chemical properties that allow the enzyme to recognize a specific substrate and bind with it. The basic characteristics of the active sites include: Very small size compare to the entire enzyme molecule i.e. the active site of an enzyme occupies only a very small portion of the enzyme molecule. The active site is also called the binding site or catalytic site. That is the substrate binds with the enzyme at the active site. 40  Active site Its sequences of amino acid fold to form three dimensional structure which is found as a cleft or a cervices on the enzyme molecule. – Enzymes must be larger than their active sites so that the site chains responsible for binding and for catalyzing reactions can be juxtaposed appropriately into three dimensions such that the active site is always found in a cleft or crevice. The active site is also called the binding site or catalytic site. That is the substrate binds with the enzyme at the active site. It has two distinct functions binding substrate(s) and catalysis. 41  When the enzyme and substrate are connected, it is known as enzyme-substrate complex. The enzyme-substrate complex is central to the action of enzymes. Enzyme-substrate complex The enzymatic reactions takes place by binding of the substrate with the active site of the enzyme molecule by several weak 97 bonds.  ENZYME NOMENCLATURE Nomenclature is a system of names or terms, or the rules for forming names. The principles of naming vary from discipline to discipline. The act of assigning names to enzymes is called enzyme nomenclature. The earliest enzymes discovered and studied were few and were given trivial or nondescriptive names as they were discovered 43  Enzyme nomenclature cont… Example: – Rennin- Curding of milk to start Cheese-making processes – Pepsin- hydrolyses protein at acidic pH. – Trypsin- hydrolyses protein at mild alkaline pH – Chymotrypsin – Catalase etc These names are historical and gives no idea of the source, function or reaction catalyzed by the enzyme. No direct relationship to the substrate or reaction type 44  Enzyme nomenclature Enzymes are named Suffix of an enzyme- ase according to the –Lactase, amylase, lipase or protease type of reaction they Denotes an enzyme catalyze and/or their substrate Some digestive enzymes have the suffix –in –Pepsin, trypsin& chymotrypsin Substrate= the reactant upon which the specific Prefix denotes the type of reaction the enzyme enzyme acts catalyzes –Enzyme physically binds –Oxidase: redox reaction to the substrat –Hydrolase: Addition of water to break one component into two parts Substrate identity is often used together with the reaction type –Pyruvate carboxylase, lactate dehydrogenase  Enzyme nomenclature cont… The nomenclature was later improved upon by adding the suffix -ase to the name of the substrate with which the enzyme functions i.e. react with. – In most cases, enzyme names end with –ase Example: Substrate Enzyme α-amylose α-amylase Lactose Lactase Maltose Maltase Lipid(fat) Lipase Urea Urease Cellobiose Cellobiase Fumarate Fumarase 46  Enzyme nomenclature cont… In the 1950’s the increasing amounts of known enzymes were causing confusion. No official naming system for enzymes. The IUBMB created the International Commission on Enzymes in 1956 to deal with enzyme nomenclature Later replaced with the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) 47  Enzyme nomenclature cont… IUBMB has recommended system of nomenclature for enzymes & according to them each enzyme is assigned with two names: Trivial name (common name, recommended name). Systemic name ( official name ). Systemic name Each enzyme is characterized by a code no.called Enzyme Code no. or EC number and contain four Figure (digit) separated by a dot. e.g. EC m. n. o. p First digit represents the class; Second digit stands for subclass ; Third digit stands for the sub-sub class or subgroup; Fourth digit gives the serial number of the particular enzyme in the list. e.g. EC 2.7.1.1 for hexokinase. 48  ENZYME CLASSIFICATION The most accepted classification of enzymes is based on the types of reactions that they Catalyze. Enzymes are classified into six major classes according to the reaction catalyzed (EC number Classification) 107  Enzyme specificity Enzymes are highly specific Interact with only one or a few of the substrates Specificity refers to the ability of an enzyme to discriminate between two competing substrates. Enzymes are highly specific in their action compared with chemical catalysts. Enzymes can distinguish between closely related – chemical species – D and L isomers – cis and trans isomers – Diastereomers (glucose and galactose)  Enzyme Specificity Cont… Enzymes have a high degree of specificity for their substrates and the type of reactions they catalyze. The specificity of enzymes is determined by its active site. The formation of the enzyme substrate complex can occur only if the substrate possesses groups which are in the correct three dimensional arrangement to interact with the binding groups of the active site. 51  Types of Enzyme Specificity Enzymes exhibit different types of specificity (a) Substrate specifity-this may be absolute, relative or broad: – Absolute specificity: Some enzymes act on only one substrate. Such enzymes are said to exhibit absolute specificity. For example, succinate dehydrogenase, a key enzyme of TCA cycle catalyzes only the oxidation of succinate to fumarate. – Absolute group specificity: Some other enzymes act on a very small group of substrates having the same functional group but at different rates. Such enzymes are said to exhibit absolute group specificity. For example, alcohol dehydrogenase oxidizes both ethanol and methanol which have common hydroxyl group. Similarly, hexokinase not only phosphorylates glucose but also fructose and mannose. – Relative group specificity: Some other enzymes exhibit relative group specificity where a given enzyme can act upon more than one group of substrates. For example, trypsin catalyzes the hydrolysis of both ester and amide bonds. 124  Models of Enzyme Specificity Enzymes are so specific because the active site of each enzyme has the proper – shape, – size and – charge to bind certain substrates only and to catalyze the conversion of these substrates to specific products. There are two models that explain enzyme specificity: – The Lock and Key model by Emil Fischer – The induced-fit model by Daniel Koshland 53  Enzyme-substrate binding Two models have been proposed – Lock and key binding The lock and key model of enzyme specificity proposed by Emil Fischer in 1894 holds that the active site of an unbound enzyme is complementary in shape to that of the substrate. – Induced fit binding The induced fit model proposed by Daniel Koshland in 1968 holds that the active site of an enzyme has a shape complementary to that of the substrate only after the substrate is bound to the enzyme.  Lock and key binding The enzyme has an active site that fits the exact dimensions of the substrate Page 460 Voet Biochemistry 3e © 2004 John Wiley & Sons, Inc.  Induced fit binding After the binding of substrate, the enzyme changes its shape to fit more perfectly with substrate  Induced-fit model cont… In the induced fit model, substrate does not fit precisely into a rigid active site. Instead noncovalent interactions between the enzyme and the substrate changes the structure of the active site conforming it to the shape of the substrate. The induced-fit model of enzyme action assumes that the enzyme active site: –is a flexible pocket –changes conformation to accommodate the substrate molecule. The induced-fit model replaced the lock and key model because it accounts for the flexible nature of proteins of which enzymes are. How do enzymes act/work?  ENZYME CATALYSIS The catalytic action of an enzyme is referred to as its activity, is measured by determining the increase in the reaction rate under precisely defined conditions. The catalytic ability of an enzyme is determined by the active site because it is the region on the enzyme responsible for binding substrate (and the prosthetic group if any). Every chemical reaction is characterized by an equilibrium constant which is a reflection of the difference in energy between reactants and products Generally, enzymes increase the rate or velocity, V, of many physiological reactions in biological systems by lowering the activation energy of the reactions they catalyze. 60 Enzymes decrease activation energy of a reaction It is believed that enzymes bind to their substrates and lower the energy required for activation of the reaction, the reaction occurs and the enzyme is then released unchanged to be used again. In every chemical reaction, the reactants pass through a transition state that has greater energy than that of the reactants or products alone The difference in energy between the reactants and the transition state is called the activation energy If the activation energy is available then the reaction can proceed forming products  The initial interaction of the enzyme with the substrate leads to the formation of enzyme–substrate complex (ES). Enzymes lower the activation energy by forming ES complex. ES is formed through the interactions between the enzyme and substrate. Each interaction releases a small amount of energy to stabilize the complex. These interactions combine to lower the activation energy of the reaction. The uncatalyzed reaction has a large activation energy, Ea, than the catalyzed reaction In the catalyzed reaction, the activation energy has been Lowered significantly leading to increased rate of the reaction. This provides a lower energy route for conversion of substrate to product. Enzymes lower the activation energy, but do not change the free energy required for the reaction to occur (alters the rate, but not thermodynamics).  An enzyme reduces the activation energy required for a reaction It provides an alternative transition state of lower energy called the enzyme- substrate complex and thus speeds up the reaction Enzymes decrease the activation energy but they do not alter the change in the free energy (∆G)  The effect of a catalyst on the transition state diagram of a reaction Page 477 Voet Biochemistry 3e © 2004 John Wiley & Sons, Inc.  Factors that affect enzyme activity Effect of temperature – Every enzyme has an optimal temp. for catalyzing a reaction – The rate of an enzyme reaction initially increases with rise in temperature – At high temp. enzymes are denatured and become inactive – In humans most enzyme have an optimal temp. of 37oC  Factors that affect enzyme activity Effect of pH – Effect of pH on the ionizable groups in the active site of enzyme or in the substrate affect catalysis – Every enzyme has an optimal pH for catalyzing a reaction – Most enzymes have highest activity between pH 6 and pH 8 – Pepsin has highest activity at pH 2  Effect of pH on the initial rate of the reaction catalyzed by most enzymes (the bell-shaped curve) Page 487 Voet Biochemistry 3e © 2004 John Wiley & Sons, Inc.  Factors that affect enzyme activity Effect of [E] and [S] – The reaction velocity increases initially with increasing [S] – At low [S], the reaction rate is proportional to [S] – Further addition of substrate has no effect on enzyme velocity (v) – The rate of an enzyme reaction is directly proportional to the conc. of enzyme if the substrate concentration [S] is higher than enzyme  Enzyme kinetics The model of enzyme kinetics was first proposed by Michaelis and Menten in 1913 and later modified by Briggs and Haldane The Michaelis Menten equation describes the relationship of initial rate of an enzyme reaction to the [S]  Enzyme Kinetic Enzyme kinetics offers a wealth of information on the mechanisms of enzyme catalysis and on the interactions of enzymes with ligands, such as substrates and inhibitors. Enzyme kinetics is the study of the rates of enzyme-catalyzed reactions. Each time vary or increase the concentration of substrate, we must measure the reaction velocity of the enzyme. The reaction velocity describes the rate at which the enzyme operates on the substrate.