Enzymes

1. Enzymes 1 • It's true hard work never killed anybody, but I figure, why take the chance? Ronald Reagan

2. ENZYMES • Thermodynamic principles can be used to indicate whether or not a reaction can take place spontaneously • They do not, however, provide information about the rate at which a reaction will proceed • Most biochemical reactions proceed so slowly at physiological temperatures that catalysis is essential for the reactions to proceed at a satisfactory rate in the cell • At temperatures above absolute zero, all molecules possess vibrational energy which increases as the molecules are heated • As the temperature rises, vibrating molecules are more likely to collide • A chemical reaction occurs when colliding molecules possess a minimum amount of energy called the activation energy • Not all collisions result in chemical reactions because only a fraction of the molecules have sufficient energy or the correct orientation to react

3. Another way of increasing the likelihood of collisions, thereby the formation of product, is to increase the concentration of reactants • In living systems, however, elevated temperatures may harm delicate biological structures and reactant concentrations are usually quite low • The preferred catalysts in living systems, therefore, are enzymes, most of which are proteins (except for ribozymes which are RNA) • Enzymes can increase the rate of a reaction by several orders of magnitude • Enzymes do their job by decreasing activation energy, thereby increasing the percentage of substrate molecules that have sufficient energy to react • Indeed, in the absence of enzymes, life as we know it would not be possible

4. Enzymes do not affect ΔG (the position of the equilibrium) but speed up its attainment. The concentrations of substrate and product at equilibrium are not changed The Effect of Catalysis on the Activation Energy of a Reaction

5. The Basic Features of Enzymes • Catalytic power • Enzymes accelerate reaction rates as much as 1016 over uncatalyzed levels, which is far greater than any synthetic catalysts can achieve • And enzymes accomplish these astounding feats in dilute aqueous solutions under mild conditions of temperature and pH • Specificity • The action of enzymes is usually very specific. This applies not only to the type of reaction being catalyzed (reaction specificity), but also to the nature of the substrates that are involved (substrate specificity) • Enzymes with low reaction specificity and low substrate specificity are very rare • Intimate interaction between an enzyme and its substrates occurs through molecular recognition based on structural complementarity

6. Such mutual recognition is the basis for specificity. The specific site on the enzyme where substrate binds and catalysis occurs is called the active site • Regulation • Although the enormous catalytic potential is essential, it does pose a problem: if it was not regulated, all reactions in a cell would rapidly reach equilibrium and, once again, life as we know it would not be possible • Regulation of enzyme activity is achieved in a variety of ways, ranging from controls over the amount of enzyme protein produced by the cell to more rapid, reversible interactions of the enzyme with metabolic inhibitors and activators • There should be a system for the classification and naming of the several enzymes present in the cell

7. Enzyme Nomenclature • The commonly used names for most enzymes describe the type of reaction catalyzed, followed by the suffix –ase • For example, dehydrogenases remove hydrogen atoms, proteases hydrolyze proteins and isomerases catalyze rearrangements • Modifiers may precede the name to indicate the substrate (lysyloxidase), the source of the enzyme (pancreatic ribonuclease), its regulation (hormone-sensitive lipase) or a feature of its mechanism of action (cysteine protease) • Where needed, alphanumeric designators are added to identify multiple forms of an enzyme (eg, RNA polymerase III; protein kinase Cβ). • To address ambiguities, the International Union of Biochemists (IUB) developed an unambiguous system of enzyme nomenclature

8. In this system, each enzyme has a unique name and code number that identify the type of reaction catalyzed and the substrates involved • Although common names for many enzymes remain in use, all enzymes now are classified and formally named according to the reaction they catalyze • Six classes of reactions are recognized. Within each class are subclasses, and under each subclass are subsubclasses within which individual enzymes are listed • Classes, subclasses, subsubclasses and individual entries are each numbered, so that a series of four numbers serves to specify a particular enzyme –Enzyme Commission (EC) Number • A systematic name, descriptive of the reaction, is also assigned to each entry

9. Classes of Enzymes • Oxidoreductases • Oxidative reactions remove electrons, usually one or two electrons per molecule of substrate, while reductive reactions accomplish the converse • Oxidoreductases transfer electrons from one compound to another, thus changing the oxidation state of both substrates • In many oxidation reduction reactions, hydrogen is transferred along with electrons • In other reactions, a molecule or atom of oxygen could be transferred to a substance; electrons could also be transferred to oxygen • Transferases • Transferases catalyze reactions in which a functional group is transferred from one compound to another • Commonly transferred functional groups include phosphate, amino, methyl

10. Hydrolases • Cleave carbon-oxygen, carbon-nitrogen, carbon-sulfur,… bonds by adding water across the bond • Digestive enzymes are hydrolases • Lyases • Cleave carbon-oxygen, carbon-nitrogen, carbon-sulfur,… bonds but do so without addition of water and without oxidizing or reducing the substrates • Double bonds either arise or disappear through the action of lyases • Isomerases • Catalyze intramolecular rearrangements of functional groups that reversibly interconvert optical or geometric isomers • When an isomerase catalyzes an intramolecular rearrangement involving movement of a functional group, it is called a mutase; what is a racemase?

11. Ligases • Catalyze biosynthetic reactions that form a covalent bond between two substrates. • Ligases differ from lyases in that they utilize the energy obtained from cleavage of a high-energy bond to drive the reaction. The molecule with the high-energy bond is usually ATP • Ligases are sometimes known as synthetases and lyases , synthases • Because the systematic names were frequently cumbersome and the numbers difficult to memorize, the EC also proposed that a single recommended (trivial) name should be retained (or invented) for each enzyme • For example, the enzyme catalyzing the reaction: • ATP+AMP<----->2ADP • bears the systematic name ATP : AMP phosphotransferase, • the number EC 2.7.4.3 and the recommended name, • adenylate kinase; the earlier name, myokinase, was forsaken

16. Cofactors • Many enzymes carry out their catalytic function relying solely on their protein structure • Many others require non-protein components, called cofactors • Cofactors may be metal ions or organic molecules referred to as coenzymes • Cofactors, because they are structurally less complex than proteins, tend to be stable to heat. Typically, proteins are denatured under such conditions • Usually coenzymes are actively involved in the catalytic reaction of the enzyme, often serving as intermediate carriers of functional groups in the conversion of substrates to products • In many cases, a cofactor is firmly associated with its enzyme, through covalent or non-covalent bonds. Such tightly bound cofactors are referred to as prosthetic groupsof the enzyme

17. Enzymes in which metal ions serve as prosthetic groups are called metalloenzymes. Enzymes that require a non-bound metal ion cofactor are termed metal-activated enzymes • The catalytically active complex of protein and prosthetic group is called the holoenzyme. The protein without the prosthetic group is called the apoenzyme; it is catalytically inactive • Many coenzymes are vitamins or contain vitamins as part of their structure • Vitamins are small organic molecules that are not synthesized in the body and are therefore essential dietary nutrients • The vitamins that are coenzyme precursors or coenzymes include all the water-soluble B vitamins, vitamin C and the fat-soluble vitamin K • Many coenzymes contain, in addition, the adenine, ribose and phosphoryl moieties of AMP or ADP

18. Coenzymes are typically modified by certain reactions and are then converted back to their original forms by other enzymes; small amounts of these substances can be used repeatedly • Vitamin B1: Thiamine • It is the precursor of thiamine pyrophosphate (TPP), a coenzyme involved in reactions where bonds to carbonyl carbons (aldehydes or ketones) are synthesized or cleaved

19. Niacin (nicotinic acid): Vitamin B3 • Nicotinamide is an essential part of two important coenzymes: nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) • The reduced forms of these coenzymes are NADH and NADPH • The nicotinamide coenzymes (also known as pyridine nucleotides) are electron carriers. They play vital roles in a variety of enzyme–catalyzed oxidation–reduction reactions • NAD+ is an electron acceptor in oxidative (catabolic) pathways and NADPH is an electron donor in reductive (biosynthetic) pathways • These reactions involve direct transfer of hydride anion (H:) either to NAD(P) + or from NAD(P)H • The hydride anion contains two electrons, and thus NAD + and NADP + act exclusively as two-electron carriers

20. The C-4 position of the pyridine ring, which can either accept or donate hydride ion, is the reactive center of NAD + and NADP+ • Humans can synthesize some amount of niacin from tryptophan. However, if dietary intake of tryptophan is low, nicotinic acid is required for optimal health • Nicotinic acid, which is beneficial to humans and animals, is structurally related to nicotine, a highly toxic tobacco alkaloid • In order to avoid confusion of nicotinic acid and nicotinamide with nicotine itself, niacin was adopted as a common name for nicotinic acid

21. The Structures and Redox States of the Nicotinamide Coenzymes

22. Riboflavin: Vitamin B2 • Is a precursor of both riboflavin 5-phosphate, also known as flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) • The name riboflavin is a synthesis of the names for the molecule’s component parts, ribitol and flavin • The flavins have a characteristic bright yellow color and take their name from the Latin flavus for “yellow” • The oxidized form of the isoalloxazine structure absorbs light around 450 nm (in the visible region) and also at 350 to 380 nm • The color is lost, however, when the ring is reduced or “bleached” • Similarly, the enzymes that bind flavins, known as flavoenzymes, can be yellow, red, or green in their oxidized states. These enzymes also lose their color on reduction of the bound flavin group

23. The Structures of FAD and FMN

24. Flavin coenzymes can exist in any of three different redox states: fully oxidized flavin is converted to a semiquinone by a one-electron transfer; a second one-electron transfer converts the semiquinone to the completely reduced dihydroflavin • The three different redox states allow flavins to participate in one-electron transfer and two-electron transfer reactions The Oxidation States of FAD and FMN

25. Panthotenic Acid: Vitamin B5 • Makes up one part of a complex coenzyme called coenzyme A (CoA) • Pantothenic acid is also a constituent of acyl carrier protein (ACP) • Coenzyme A consists of 3,5-adenosine bisphosphate joined to 4-phosphopantetheine in a phosphoric anhydride linkage • As was the case for the nicotinamide and flavin coenzymes, CoA also contains an adenine nucleotide moiety • CoA and ACP are involved in the activation and transfer of acyl groups • The functions of CoA are mediated by the reactive sulfhydryl group on CoA, which forms thioester linkages with acyl groups

26. The Structure of Coenzyme A

27. Pyridoxine: Vitamin B6 • The biologically active form of vitamin B6 is pyridoxal-5-phosphate (PLP) • PLP participates in the catalysis of a wide variety of reactions involving amino acids, including transaminations, decarboxylations, racemizations and eliminations • PLP is found as a prosthetic group attached to enzymes through a Schiff base formed between its aldehyde group and the ε-amino group of a lysine residue

28. PLP Attached to an Enzyme The Tautomeric Forms of PLP

29. Biotin : Vitamin B7 • Acts as a mobile carboxyl group carrier in a variety of enzymatic carboxylation reactions • In each of these, biotin is bound covalently to the enzyme as a prosthetic group via the ε-amino group of a lysine residue on the protein • The biotin-lysine function is referred to as a biocytin residue The result is that the biotin ring system is tethered to the protein by a long, flexible chain The Biocytin Complex

30. Folic Acid: Vitamin B9 • Folic acid derivatives (folates) are acceptors and donors of one-carbon units for all oxidation levels of carbon except that of CO2 (where biotin is the relevant carrier) • The active coenzyme form of folic acid is tetrahydrofolate (THF) • THF is formed via two successive reductions of folate by dihydrofolate reductase Folic Acid

31. THF • Cyanocobalamin: Vitamin B12 • Cyanocobalamin is converted in the body into two coenzymes: the predominant coenzyme form is 5-deoxyadenosylcobalamin, but smaller amounts of methylcobalamin also exist • The corrin ring, with four pyrrole groups, is similar to the heme prophyrin ring, except that two of the pyrrole rings are linked directly; iron is substituted by cobalt • There are two reactions in the body in which vitamin B12 is known to participate: a molecular rearrangement and a methyl transfer

32. Vitamin B12 and its Coenzyme Forms

33. Ascorbic acid: Vitamin C • Has the simplest chemical structure of all the vitamins; and the coenzyme form is the vitamin itself • Ascorbic acid functions as an electron carrier. It is a strong reducing agent • It is used in the regeneration of the active form of enzymes and antioxidants (-) 2H. (+) H. (-) H. (+) 2H.

34. The Lipid-Soluble Vitamins • Vitamin K is the only fat-soluble vitamin with coenzyme role • The Vitamin A group • Vitamin A or retinol often occurs in the form of esters, called retinyl esters. The aldehyde form is called retinal or retinaldehyde • Retinol can be absorbed in the diet from animal sources or synthesized from β-carotene from plant sources • The aldehyde group of retinal forms a Schiff base with a lysine on opsin, to form light-sensitive rhodopsin • The Vitamin D group • The two most prominent members of the vitamin D family are ergocalciferol (vitamin D2) in plants and cholecalciferol (vitamin D3) in animals • Cholecalciferol is produced in the skin of animals by the action of ultraviolet light (sunlight, for example) on its precursor molecule, 7-dehydrocholesterol

35. Rhodopsin

36. Because humans can produce vitamin D3, “vitamin D” is not strictly speaking a vitamin at all • Retinol and cholecalciferol are actually prohormones(precursors of hormones) that regulate transcription of DNA, and thus gene expression • Tocopherol: Vitamin E • α-tocopherol is a potent antioxidant; and once it has been oxidized, it can be regenerated by vitamin C

37. Naphthoquinone: Vitamin K • Clotting factors such as thrombin undergo a post-translational modification that involves the carboxylation of glutmate residues • γ-carboxyglutamyl residues are effective in the coordination of calcium, which is required for the coagulation process • The enzyme responsible for this modification, glutamylcarboxylase, requires vitamin K for its activity The Structure of the K Vitamins

38. Not all coenzymes are derived from vitamins • Tetrahydrobiopterin (BH4), the coenzyme for hydroxylation reactions of aromatic amino acids is synthesized in the body from GTP (guanosinetriphosphate) • Lipoic acid is a coenzyme used to couple acyl transfer with electron transfer • Lipoic acid exists as a mixture of two structures: a closed-ring disulfide form and an open–chain reduced form. Oxidation–reduction cycles interconvert these two species • Lipoic acid is found attached with a lysine residues on enzymes • Metal ions, which have a positive charge, contribute to the catalytic process by acting as electrophiles . They assist in binding of the substrate or they stabilize developing anions in the reaction. They can also accept and donate electrons in oxidation-reduction reactions

39. Examples of metal ions and the enzymes they function with include: • Zn2+: alcohol dehydrogenase, carbonic anhydrase • Mg2+: ATP-dependent reactions such as hexokinase • Fe3+and Cu2+:components of the enzymes of the mitochondrial electron transport chain to the ultimate electron acceptor, oxygen The Different forms of Lipoic Acid

40. How Do Enzymes Work? • There are three characteristics of enzymes that form the basis of most of their properties: • The Active Site • In an enzyme, folding brings together amino acids, most of which are not adjacent in the primary sequence, so that some amino acids form a three-dimensional structure that binds with the substrate to form the enzyme-substrate complex • This complex results in catalysis • The remainder of the amino acids in the enzyme are involved in maintenance of the three-dimensional structure of the enzyme, attaching the enzyme molecule to intracellular structures (e.g. membranes) or in binding molecules (e.g. allosteric effectors) that regulate the activity of the enzyme • The Enzyme-Substrate Complex • Enzymes bind substrates to produce an enzyme-substrate complex as follows:

41. Amino Acid Side-Chain Groups Involved in Binding NAD+ at the Active Site of an Enzyme

42. k1 • E+S⇌ES • Weak bonds, generally non-covalent ones, are involved in formation of the complex, so that the reaction is readily reversed • The rate of the forward reaction is given by the concentration of substrate multiplied by the rate constant k1, and rate of the reverse reaction is given by the concentration of the product multiplied by the rate constant k2 • The dissociation constant for the ES complex is k2/k1 • This is analogous to the formation of other complexes: for example receptor-hormone complex; receptor-neurotransmitter complex; antibody-antigen complex • Formation of the enzyme-substrate complex can occur only if the substrate possesses groups that are in the correct three-dimensional orientation to interact with the binding groups in the active site k2

43. A ‘lock and key’ analogy (Emil Fischer) has been widely used to explain specificity but it is inadequate because the formation of the enzyme-substrate complex involves more than a steric complementarity between enzyme and substrate • Enzymes are highly flexible, conformationally dynamic molecules, and many of their remarkable properties, including substrate binding and catalysis, are due to this flexibility • Realization of the conformational flexibility of proteins led Daniel Koshland to hypothesize that the binding of a substrate by an enzyme is an interactive process • The shape of the enzyme’s active site is actually modified upon binding S, in a process of dynamic recognition between enzyme and substrate called ‘induced fit’ • Substrate binding alters the conformation of the protein, so that the protein and the substrate “fit” each other more precisely. The conformation of the substrate also changes as it adapts to the conformation of the enzyme

44. The Transition State • In a chemical reaction, one stable arrangement of atoms (the substrate) is converted to another (the product) • As this change proceeds, the atoms pass through an unstable arrangement, known as the transition state, which can be thought of as the ‘halfway house’ between the substrates and the products • The relevance of the transition state to kinetics is that the rate of the overall reaction depends on the number of molecules in this state: the more molecules in the transition state, the greater is the rate • The role of an enzyme is to increase the number of molecules in this state • Enzymes increase the number of molecules in the transition state through one or more of five mechanisms

45. The Energy Level and the Structural Feature of the Transition State

46. Mechanisms of Enhancing the Rate of a Reaction • Since the transition state possesses the least stable electron distribution, an agent capable of supplying or withdrawing electrons to or from stable parts of a substrate in order to destabilize it, accelerates the rate of a reaction • General acid base catalysis • Addition of a proton from an acid to a molecule can cause an electron to be withdrawn from one part of the molecule to the part which binds the proton • A base removes a proton from a molecule which will also cause electron shifts • If these shifts favor the formation of the transition state, the rate of the reaction increases • The active sites of enzymes possess side-chain groups of amino acids that act as acids or bases

47. The contribution of these groups is greatly enhanced if they act in a concerted manner so that, as an electron is withdrawn from one part of the substrate, another is donated to a different part • This is possible only when the relevant groups in the active site are held in precisely the correct orientation so as to interact in this way with the substrate • One of the more versatile side-chains in this respect is the imidazole group of histidine. In one environment it can act as an acid whereas, in another environment, the same group can act as a base • This can occur with two histidines in the same active site • Acid base catalysis occurs on the vast majority of enzymes. In fact, proton transfers are the most common biochemical reactions • Specific acid base catalysis occurs when only the protons and hydroxyls present in solution are used

48. Amino Acids Involved in Acid Base Catalysis

49. Covalent catalysis (formation of an intermediate) • Most enzymes bind their substrates in a non-covalent manner but, for those that do bind covalently, the intermediate must be less stable than either substrate or product • Many of the enzymes that involve covalent catalysis are hydrolytic enzymes; these include proteases, lipases, phosphatases and also acetylcholinesterase • A number of amino acid side chains, including all those that participate in acid base catalysis and the functional groups of some coenzymes can serve as nucleophiles in the formation of covalent bonds with substrates. These covalent complexes always undergo further reaction to regenerate the free enzyme An Example of Covalent Catalysis

50. An Example of Covalent Catalysis • Metal Ion Catalysis • Metals, whether tightly bound to the enzyme or taken up from solution along with the substrate, can participate in catalysis in several ways • Ionic interactions between an enzyme-bound metal and a substrate can help orient the substrate for reaction or stabilize charged reaction transition states • Metals can also mediate oxidation-reduction reactions by reversible changes in the metal ion’s oxidation state • Nearly a third of all known enzymes require one or more metal ions for catalytic activity