Chapter 8 Enzyme Catalysis

1. Chapter 8 Enzyme Catalysis Homework II (cont’d): Chapter 8, Problems 4, 5, 7, 8, 9, 11 (the diagonal line with “Slope” is the blue line), 12, 16, 17 Due October 25 (Wed).

2. 1. Enzymes were among the first biological macromolecules to be studied chemically 1.1 Much of the early history of biochemistry is the history of enzyme research. 1.1.1 Biological catalysts were first recognized in studying animal food digestion and sugar fermentation with yeast (brewing and wine making). 1.1.2 Ferments (i.e., enzymes, meaning in “in yeast”) were thought (wrongly) to be inseparable from living yeast cells for quite some time (Louis Pasteur)

3. 1.1.3 Yeast extracts were found to be able to ferment sugar to alcohol (Eduard Buchner, 1897, who won the Nobel Prize in Chemistry in 1907 for this discovery). 1.1.4 Enzymes were found to be proteins (1920s to 1930s, James Sumner on urease and catalase, “all enzymes are proteins”, John Northrop on pepsin and trypsin, both shared the 1946 Nobel Prize in Chemistry). 1.1.5 Almost every chemical reaction in a cell is catalyzed by an enzyme (thousands have been purified and studied, many more are still to be discovered!) 1.1.6 Proteins do not have the absolute monopoly on catalysis in cells. Catalytic RNA were found in the 1980s (Thomas Cech, Nobel Prize in Chemistry in 1989).

4. 2. The most striking characteristics of enzymes are their immense catalytic power and high specificity. 2.1 Enzymes accelerate reactions by factors of at least a million. 2.1.1 Most reactions in biological systems do not occur at perceptible rates in the absence of enzymes. 2.1.2 The rate enhancements (rate with enzyme catalysis divided by rate without enzyme catalysis) brought about by enzymes are often in the range of 107 to 1014) 2.1.3 For carbonic anhydrase, an enzyme catalyzing the hydration of CO2 (H2O + CO2 HCO3- + H+), the rate enhancement is 107 (each enzyme molecule can hydrate 105 molecules of CO2 per second!)

5. 2.2 Enzymes are highly specific both in the reaction catalyzed and in their choice of substrates (i.e., reactants). 2.2.1 An enzyme usually catalyzes a single chemical reaction or a set of closely related reactions (side reactions leading to the wasteful formation of by-products rarely occur). 2.2.2 Enzymes exhibit various degrees of specificity in accord with their physiological functions (what of the following?): Low specificity: some peptidases, esterases, and phosphatases. Intermediate specificity: hexokinase, alcohol dehydrogenases, trypsin. Absolute or near absolute specificity: Many enzymes belong to this group, and in extreme cases, stereochemical specificity is exhibited (i.e., enantiomers are distinguished as substrates or products).

6. 2.3 Most enzymes are proteins. 2.3.1 Some enzymes require no other chemical groups other than their amino acid residues for activity. (e.g.) 2.3.2 Other enzymes require additional chemical components called prosthetic groups (covalently bound) (or cofactors). 2.3.3 Prosthetic groups could be inorganic metal ions (e.g., Fe2+, Mg2+, Mn2+, Zn2+) or complex organic or metalloorganic molecules called coenzymes. 2.3.4 A complete catalytically active enzyme (including its prosthetic group) is called a holoenzyme. 2.3.5 The protein part of an enzyme (without its prosthetic group) is called the apoenzyme.

7. 2.3.6 Coenzymes often function as transient carriers of specific (functional) groups during catalysis. 2.3.7 Many vitamins, organic nutrients required in small amounts in the diet, are precursors of coenzymes.

11. 3. Enzymes are classified by the reactions they catalyze 3.1 Trivial names are usually given to enzymes. 3.1.1 Many enzymes have been named by adding the suffix “-ase” to the name of their substrate or to a word or phrase describing their activity (type of reaction). 3.2 Enzymes are categorized into six major classes by international agreement. 3.2.1 The six major classes include Oxidoreductases: catalyzing oxidation-reduction reactions. Transferases: catalyzing the transfer of a molecular group from one molecule to another.

12. 3.2.1 (cont’d) Hydrolases: catalyzing the cleavage by the introduction of water. Lyases: catalyzing reactions involving removal of a group to form a double bond or addition of groups to double bonds. (e.g.?). Isomerases: catalyzing reactions involving intramolecular rearrangements. Ligases: catalyzing reactions joining together two molecules.

13. 3.3 Each enzyme is given a systematic name which identifies the reaction catalyzed (e.g., hexokinase is named as ATP:glucose phosphotrasferase). 3.4 Each enzyme is assigned a four-digit number with the first digit denoting the class it belongs, the other three further clarifications on the reaction catalyzed.

15. 4. Enzymes, like all other catalysts, does not affect reaction equilibria, only accelerate reactions. 4.1 Equilibrium constant (Keq’) of a reaction is related to the free energy difference between the ground states of the substrates and products (Go’)  Go’ = -RTlnKeq’ Enzyme catalysis does not affect  Go’, thus not Keq’.

16. 4.2 The rate constant of a reaction (k) is related to the free energy difference between the transition state and the ground state of the substrate (G‡) 4.2.1 Transition state is a fleeting molecular moment (not a chemical species with any significant stability) that has the highest free energy during a reaction. 4.2.2 An enzyme increases the rate constant of a reaction (k) by lowering its G‡. 4.2.3 The combination of a substrate and an enzyme creates a new reaction pathway whose transition state energy is lower than that of the reaction in the absence of energy.

20. 5. Formation of an enzyme-substrate complex is the first step in enzyme catalysis. 5.1 Substrates are bound to a specific region of an enzyme called the active site. 5.1.1 Much of the catalytic power of enzymes comes from their bringing substrates together in favorable orientations in enzyme-substrate (ES) complexes. (mutations that affect the on-rate, the off-rate, kcat, …etc). 5.1.2 Most enzymes are highly selective in their binding of substrates. 5.1.3 The active site usually takes up a relatively small part of the total volume of an enzyme. 5.1.4 The active site is a three-dimensional entity formed by groups that come from different parts of the linear amino acid sequence.

21. 5.1.5 Substrates are bound to enzymes by multiple weak (noncovalent) attractions. 5.1.6 Active sites are clefts or crevices with a generally nonpolar character (polar residues, when present in the active site, usually participate in the catalytic processes, thus called catalytic groups) (or specificity of binding). 5.1.7 The active sites of some unbound enzymes are complementary in shape to those of their substrates (the lock-and-key metaphor, Emil Fisher). 5.1.8 In many enzymes, the active sites have shapes complementary to those of their substrates only after the substrates are bound (the induced fit, Daniel Koshland).

22. 5.2 The existence of ES complexes has been shown in a variety of ways. 5.2.1 The saturation effect: at a constant concentration of an enzyme, the reaction rate increases with increasing substrate concentrations until a Vmax is reached. 5.2.2 ES complexes have been directly observed by electron microscopy and X-ray crystallography.

24. Dihydrofolate reductase, NADP+ (red), tetrahydrofolate (yellow) The lack of perfect complementarity is important to enzymatic catalysis (induced fit) (not evident in this figure).

25. 6. Binding energy is the major source of free energy used by enzymes to lower the activation energies of reactions. 6.1 Binding energy (Gb) is the energy derived from enzyme-substrate interaction. 6.1.1 Formation of each weak interaction in the ES complex is accompanied by a small release of free energy. 6.1.2 Weak interactions are maximized when the substrate is converted to the transition state. 6.1.3 The weak interactions that are formed only in the transition state are those that make the primary contribution to catalysis: Transition state theory. In another words, the enzyme is evolved (“designed”) to bind the transition state structure.

32. 6.1.4 Catalytic antibodies can be formed by using transition state analogs as immunogens (predicted by William Jencks in 1969 and confirmed by Richard Lerner and Peter Schultz in 1986). Problems: specific chemical reaction, lack of substrate specificity. E.g. Cutting DNA independent of or relative insensitive to the flanking sequence. 6.2 The summation of the unfavorable (positive) G‡ and the favorable (negative) Gb results in a lower net activation energy. 6.2.1 The requirement for multiple weak interactions to drive catalysis is one reason why enzymes (and some coenzymes) are so large. Therefore, larger Gb.

33. 6.3 Catalysis and specificity arise from the same phenomenon. 6.3.1 Catalysis refers to the acceleration of the reaction due to the involvement of enzymes. 6.3.2 Specificity refers to the ability of an enzyme to discriminate between two competing substrates. 6.3.3 The same binding energy that provides energy for catalysis also makes the enzyme specific. (Gb-transition-state + Gb-substrate-general + Gb-substrate-specific) But they are not always separable. It is intellectually useful to make distinctions among these binding energies, attributable to a particular group of atomic interactions. They are often used in the combined interpretation of kinetic, biochemical, genetic, and structural data.

34. 6.4 Binding energies can be used to overcome various energy barriers that exist during catalysis. 6.4.1 Binding energy holds the substrates in the proper orientation to react, thus overcome entropy reduction (substrate recognition and complex formation). 6.4.2 Formation of weak bonds between substrate and enzyme also results in the unfavorable desolvation of the substrate (requiring a binding energy of what form? Nonspecific?). 6.4.3 Binding energy involving weak interactions formed only in the reaction transition state helps to compensate thermodynamically for any strain or distortion that substrate must undergo to react (to break or form a bond).

35. The interactions from the added groups contribute largely to the stabilization of the transition state. Moreover, the rate can be affected greatly by the interactions physically remote from the covalent bond broken.

36. 6.4.4 Weak interactions between substrates and enzymes may generate conformational changes on the enzyme, a phenomenon called induced fit. 6.4.5 Induced fit may serve to bring specific functional groups on the enzyme into the proper orientation and position (alignment) to catalysis (need to overcome the entropy increase). 6.4.6 Modern approaches combine multiple theoretical (e.g., computational) and experimental (e.g., mutagensis) approaches to the studies of enzymes.

37. Reactions of an ester with a carboxylate group to form an anhydride

40. 7. Properly positioned catalytic functional groups aid bond cleavage and formation during enzyme catalysis. 7.1 The active sites of some enzymes contain amino acid functional groups that can participate in the catalytic process as proton donors or proton acceptors--general acid-base catalysis. 7.1.1 Many biochemical reactions involve the formation of unstable charged intermediates that tend to break down rapidly to their constituent reactant species.

41. 7.1.2 Charged intermediates can often be stabilized by transferring protons to or from the substrate or intermediate to form a species that breaks down to products more readily than to reactants. Catalysis here means the facilitated (coordinated, aligned) proton transfer. 7.1.3 General acid-base catalysis can provide a rate enhancement on the order of 102 to 105. 7.2 Some enzymes accelerate reactions by forming transient covalent intermediates with the substrates--covalent catalysis. 7.2.1 Amino acid side chains (e.g., Ser) and prosthetic groups can function as nucleophiles in forming covalent intermediates with substrates. ( leading to enzyme conformational changes)

43. 7.2.2 The new pathway of reaction must have a lower activation energy than the uncatalyzed one. 7.2.3 Free enzyme is always regenerated at the end of the reaction. 7.3 Metal ions can participate in catalysis in several ways. 7.3.1 Metal ions can be tightly bound to the enzyme or taken up from the solution along with the substrate. 7.3.2 Metal ions (bound to the enzymes) can help orient a substrate or stabilize charged reaction transition states. 7.3.3 Metal ions can help activate substrates (how? By polarizing the bond or creating a better nucleophile?)

44. 7.3.4 Metal ions can mediate oxidation-reduction reactions by reversibly change their oxidation states (electron donor and acceptor). 7.3.5 Many enzymes have metal ions in their active centers playing important roles in catalysis. 7.3.6 (Noncatalytic function) Metal ions can also have structural purposes (e.g., Ca2+ binding leads to conformational changes of calmodulin, EF-hand, etc.). Regulation of enzymes is done through regulation of their structures (conformations).

45. 7.4 An enzyme may use a combination of several catalytic strategies to bring about a rate enhancement. 7.4.1 Chymotrypsin uses both covalent catalysis and general acid-base catalysis (details?)

48. 8. Michaelis-Menton equation reflects the kinetic behavior of many enzymes 8.1 Saturation effect was observed in enzyme catalysis when plotting the initial velocity (Vo) against the substrate concentration([S]). 8.1.1 Initial velocity (Vo) was measured at the beginning of the enzyme-catalyzed reaction, when substrate concentration can be considered constant ([S] will decrease as the reaction progresses). 8.1.2 At relatively low concentrations of substrate, Voincreases almost linearly with an increase in [S].