Mechanistic Enzymology

1. MechanisticEnzymology • Recommended readingandusefulreference (1) • Richard B. Silverman (2002) The organicchemistryofenzyme-catalyzedreactions. Academic Press. • William P. Jencks (1987) Catalysis in chemistryandenzymology. Dover Publications. • Perry A. Frey, Adrian H. Hegemann (2007) Enzymaticreactionmechanisms. Oxford University Press. • Michael Sinnott (1998) Comprehensivebiologicalcatalysis. A mechanisticreference. Academic Press. • Tim Bugg (2001) An introductiontoenzymeandcoenzymechemistry. Blackwell Science. • Robert A. Copeland (2000) Enzymes. Wiley-VCH. • Alan R. Fersht (1999) Structureandmechanism in proteinscience. W. H. Freeman & Co.

2. Recommended reading and useful reference (2) • H. Gutfreund (1998) Kinetics for the life sciences. Cambridge University Press. • Irwin H. Segel (1993) Enzyme kinetics. Wiley. • Topics covered • Structure-function relationships and catalytic mechanisms of NAD(P) dependent dehydrogenases • Example of a complex, multi-step redox reaction • Enzymatic hydrolysis and synthesis of glycosides • An unusual role of pyridoxal 5‘-phosphate in catalysis • Metal cofactors in redox reactions

3. Why carbohydrates? Reactivity and rate acceleration Half life in water at 25 °C: ≈8 billion years (Acc. Chem. Res. 34, 938) Rate acceleration by hydrolytic enzymes: ≥ 1015 (Acc. Chem. Res. 33, 11) Specificity Chemical carbohydrate synthesis is usually not „green“. Applications ... from bulk to high-value chemicals.

4. Chemical and enzymatic aspects of the dehydrogenase reaction (1)

5. Chemical and enzymatic aspects of the dehydrogenase reaction (2) • Stereospecificity of hydrogen transfer to and from NAD(P)(H) A side pro-R B side pro-S A or B-side hydride transfer is a conserved feature of enzymes belonging to a certain (super)family

6. Proton NMR is a useful technique to determine the stereospecificity of a dehydrogenase of interest. Spectra are measured at the end of the reaction as shown, or in a time-resolved manner by monitoring the enzymatic reaction directly in the NMR tube.

7. S S S L L L (R-alcohol – S-alcohol)*100 (R-alcohol + S-alcohol) ee % = Chemical and enzymatic aspects of the dehydrogenase reaction (3) • Stereospecificity of hydrogen transfer to the (prochiral) carbonyl group re-side H- attack si-side H- attack S-alcohol R-alcohol

8. Chemical and enzymatic aspects of the dehydrogenase reaction (4) • The reaction is freely reversible. • Equilibrium constant of reaction is pH dependent. • NAD(P)+ + alcohol NAD(P)H + ketone + H+ • The reaction produces (or consumes) a molar equivalent of protons for each mole of NAD(P)+ reduced (or NAD(P)H oxidized). • At low buffer concentrations, the release or uptake of protons can be measured (using suitable pH indicators) and parallels the conversion of NAD(P)+ or NAD(P)H.

9. Enzymatic aspects of the dehydrogenase reaction • Kinetic mechanism of the two substrate - two product reaction • Identity of the catalytic acid-base • Mechanism of exchange of protons between the active site of the enzyme and bulk solvent • Auxiliary catalytic groups and their exact role in function • Relative timing of chemical steps of hydride and proton transfer and relatedly, the transition state structure • Rate-limiting step(s) • Residues providing stereoselectivity in the chemical reaction • Features of specificity related to physiological function and application

10. Order of binding of substrates and release of products (1) Binding of substrates by dehydrogenases is often ordered whereby coenzyme is most of the times the leading substrate and the product that dissociates last, like in the shown reaction. Enzymatic reactions involving two substrates and two products (bi-bi) can follow two other basic types of kinetic mechanism: random or Ping-Pong. The Ping-Pong mechanism is often seen when two half reactions occur. It is distinct from ordered and random mechanisms that require ternary complexes for the reaction to take place.

11. Kinetic mechanism - how do we know? (1) • Kinetic analysis using dead-end inhibitors against coenzyme and substrate is a straightforward (however, not the only) way to elucidate the kinetic mechanism of dehydrogenases. • The characteristic pattern of inhibition is evaluated whereby a competitive (C), a noncompetitive (N), and an uncompetitive (U) mode of inhibition is distinguished (using e.g. Lineweaver-Burk plot analysis). Ordered mechanism [A] varied [B] varied Inhibitor versus A (e.g. ATP for NAD(P)+) C N Inhibitor versus B (e.g. CF3CH2OH for CH3CH2OH) UC Alternative, however, characteristic patterns result for random and Ping-Pong mechanisms.

12. Kinetic mechanism - how do we know? (2) • The ordered mechanism implies formation of binary enzyme-substrate complexes (e.g. E-NAD(P)H and E-NAD(P)+) • Methods for determining ligand / substrate binding • in the steady state (F, ITC, CD, ...), yield thermodynamic data (Kd) • in the kinetic transient, yield thermodynamic + kinetic data (Kd = koff/kon) Example of binding of NAD+ to xylose reductase measured with a rapid mixing stopped flow analyzer. Quenching of the intrinsic Trp fluorescence of the enzyme is used as reporter of the binding event. Two solutions, one containing the enzyme and another containing the NAD+ are automatically mixed from two syringes and transferred into the measurement cell. The fluorescence traces are fitted with an exponential decay function, yielding rate constants (kobs). [NAD+] kobs = kon [NAD+] + koff

13. Ideally, one has estimates for each of the microscopic rate constants in the kinetic mechanism of an enzyme. Note that the Michaelis-Menten parameters that you are used to determine are functions of the the microscopic rate constants. Here, for example, kcat/KmNADH equals the expression k1/(1 + k4/k3). Protein conformational changes (k3,k4; k11,k12) are often involved in the binding and the release of NAD(P)(H). The chemical conversion takes place in steps k7 and k8. Note that most steps are physical, not chemical.

14. Structures of E and *E-NAD(P)+ explaining kinetics and vice versa Loop disordered? Loop disordered Loop ordered E-NAD(P)+ E + NAD(P)+ *E-NAD(P)+ knetiso ≈ kcat = 12 s-1 E + NAD(P)+ *E-NAD(P)+ E-NAD(P)+

15. Relative timing of chemical steps (1) Concerted or stepwise mechanism ... ... will affect negative charge development on oxygen in the transition state of the reaction

16. Relative timing of chemical steps (2) Example of a transient kinetic experiment carried out with a stopped flow reaction analyzer where the NAD+ dependent oxidation of sorbitol by a Zn2+ xylitol dehydrogenase was studied. Formation of NADH and release of protons were measured. In the pre-steady state, the release of protons was about 50% faster than production of NADH, supporting a seldom observed stepwise reaction. Note the formation of an additional equivalent of protons, which is due to deprotonation of the enzyme as sorbitol binds. Protons released NADH produced Pre-steady state control

17. Deuterium kinetic isotope effects (1) Primary effect (on bond formation and cleavage) Enzymes: VH/VD ≈ 1 - 6 Chemistry: VH/VD ≈ 10 Meaning: rate limitation by chemical step (hydride transfer), which can be masked by slow conformational steps in enzymes Secondary effect (on re-hydridisation of carbon between sp2 and sp3) Enzymes: VH/VD ≈ up to 1.3 Chemistry: VH/VD ≈ up to 1.3 Meaning and interpretation: transition state structure, from like reactants (near 1) to like products (near 1.3)

18. Deuterium kinetic isotope effects (2) Secondary effect (on proton transfer) Enzymes: VH/VD ≈ 1 - 8 Chemistry: VH/VD ≈ 10 Meaning: rate limitation by chemical step (proton transfer), which can be masked by slow conformational steps in enzymes Although useful in certain cases, experiments in D2O must be made with great care. Exchange of solvent (H2O against D2O) is an issue. Ionization equilibria are shifted in D2O or in other words, the pH optimum of an enzyme needs not be the same in H2O and D2O.

19. Deuterium kinetic isotope effects (3) Example of an experiment where the pL dependence (L = H or D) of kcat for NAD+ dependent oxidation of sorbitol by a Zn2+ sorbitol dehydrogenase from sheep liver (slSDH) was studied. At the optimum pL (≥ 8.2), the rate in H2O is significantly faster than in D2O, suggesting that proton transfer is a relatively slow step of the overall reaction. However, the observed effect is smaller than 2, indicating that other steps in the mechanism are even slower. Generally, chemical proton transfers between atoms are fast. However, small structural rearrangements in the active site required for proton transfer may become limiting.

20. Identity of the catalytic acid-base (1) • Site-directed substitution of the candidate residue (e.g. Tyr) against another residue than cannot fullfil an analogous function (e.g. Phe, Ala). • Determination of pH dependence of kinetic parameters of purified wild-type and mutant enzymes. Comparison of pH profiles for wild-type and Y51A mutant forms of xylose reductase. The wild-type enzyme shows a decrease in activity (kcat/Km) above a pK of ≈ 9 in the direction of xylose reduction and below a pK of around ≈ 7 in the direction of xylitol oxidation. The pK of almost 2 units can be explained by the effect of charge on NAD+ (which is absent in NADH) on the ionization of the catalytic acid-base. The pK of 7 is eliminated in the Y51A mutant, confirming the proposed role of the Tyr as general base catalyst for NAD+ dependent oxidation of xylitol. Wild-type (reduction) Wild-type (oxidation) log kin. parameter Y51A (oxidation) pH

21. Identity of the catalytic acid-base (2) • Chemical rescue • =to recover activity lost in a mutant by the addition of an exogenous acid / base. • = to determine dependence of rate on structural and electronic properties of the rescuing reagent. This is valuable information considering the relatively small range of possibilities offered by side chains of natural amino acids. Example of restoration of NAD+ dependent dehydrogenase activity in a Lys295-into-Ala mutant of mannitol 2-dehydrogenase from Pseudomonas fluorescens. Through addition of 2 M ethylamine, almost wild-type level of activity was recovered, despite the mutant being almost inactive in the absence of an external amine. The amine must be unprotonated for „rescue“.

22. Structure-function relationships for NAD(P)+ dependent alcohol dehydrogenases • Classification according to structural features of the domain that binds the NAD(P)+ into • Rossmann fold enzyme superfamilies • Short-chain dehydrogenases / reductases (≈ 25 kDa) • Medium-chain dehydrogenases / reductases (≈ 32 kDa) • Long-chain dehydrogenases / reductases (≥ 40 kDa) • TIM barrel enzyme superfamilies • Aldo-keto reductases (≈ 32 kDa)

23. The TIM or (/)8 barrel

24. The Rossmann fold (left domain)

25. Polyol-specific long-chain dehydrogenases - mannitol 2-dehydrogenase

26. Polyol-specific long-chain dehydrogenases Mannitol 2-dehydrogenases Mannonate oxidoreductases Arabinitol dehydrogenases Fructuronate reductases Altronate oxidoreductases Mannitol-1-phosphate dehydrogenases ... evolutionary relationships reflect substrate specificity.

27. The active site of mannitol 2-dehydrogenase with NAD+ bound

28. The active site of mannitol 2-dehydrogenase with NAD+ and mannitol bound

29. The proton channel in mannitol 2-dehydrogenase

30. Proposed mechanism of mannitol 2-dehydrogenase • Lys295 is the catalytic acid-base. • The side chains of Asn300 and Asn191 form an oxyanion-binding pocket whose role is to stabilize the partial negative charge on the oxygen of the alcohol group undergoing reaction. Mutation of either Asn by Ala slows the chemical step of hydride transfer by 3 orders of magnitude. • Double mutation of the Asn leads to an additive effect. (It is possible that two mutations are independent one of another, antagonistic, or synergistic.) • Glu292 in combination wth active site water establishes a proton relay through which Lys295 exchanges a proton with bulk solvent. Mutation of the Glu (e.g. into Gln) such that the proton relay is disrupted, leads to a „stuck proton“ and hence prevents the reaction cycle to going to completion.

31. Xylose reductase D-xylose (pyranose) 0.02 % D-xylose (free aldehyde) NAD(P)H pro-R specific reduction NAD(P)+ xylitol

32. The superfamily of aldo-keto reductases (AKR) Xylose reductase from Candida tenuis is classified as AKR2B5

33. Carbonyl group reduction in aldehydes and ketones C-C double bond reduction Endoperoxide reduction Some oxidations

34. ... is a functional dimer and adopts a TIM barrel fold

35. The active site of Candida tenuis xylose reductase

36. His114 Tyr52 Lys81 pro-R hydride from NAD(P)H Asp47 • Tyr52 is the general acid/base catalyst. • His114, through a hydrogen bond to the carbonyl oxygen, facilitates C=O bond charge separation and orbital overlap in the ternary complex. • Lys81 provides electrostatic stabilisation. • Asp47 holds the lysine side chain in place and provides connection to the bound nucleotide.

37. Substrate specificity xylose reductase for carbohydrates Ala Asp • Substrate docking: Asn310 hydrogen bonds with OH groups at C-2 and C-5. • Kinetics: interactions with C-2(R) OH are important for transition state stabilisation.

38. ... role of Asn310 in the interaction with the 2-OH of the substrate confirmed! • Catalytic efficiencies for reaction with sugars are reduced by ≈ 2 orders of magnitude in the mutants. • A substrate in which the C-2(R) hydroxy group is replaced by a hydrogen is converted with similar catalytic efficiencies by the wild type and the mutants.

39. The conformations of side chains of Asn310 and Asp310 are similar! 2.4 Å x-ray structure of N310D bound to NAD+ A conformational change of the side chain at position 310 may occur during substrate binding or after formation of the ternary complex.

40. The active site of horse liver alcohol dehydrogenase PFB ... pentafluorobenzylalcohol

41. The active site of xylitol/sorbitol dehydrogenase

42. SDR MDR Comparison of catalytic mechanisms of short-chain dehydrogenases (SDR) and medium-chain dehydrogenases (MDR)

43. Stacking of the nicotinamide ring with protein residues in the active site: xylose reductase

44. Stacking of the nicotinamide ring with protein residues in the active site: mannitol 2-dehydrogenase

45. Two-step oxidation catalyzed by UDP-glucose dehydrogenase + H+ + 2 H+

46. Reaction in three steps: Alcohol dehydrogenase: oxidation of primary OH at C-6 to yield an aldehyde (factor: general base catalysis to proton abstraction from the hydroxyl group) Aldehyde dehydrogenase: oxidation of aldehyde via the intermediate of thioester (factors: nucleophilic catalysis; electrostatic stabilisation of a tetrahedral oxyanion intermediate) Hydrolase: Hydrolysis of the thioester to yield the acid product (factor: general base catalysis to attack of water).

47. Asp280 Activation of nucleophile in step 2, via H2O Thr132 Activation of nucleophile in step 2, via H2O Asn224 Oxyanion stabilisation in step 2 Cys276 catalytic nucleophile in step 2 Lys220 catalytic base in step 1 Glu161 catalytic base in step 3 UDP-a-D-Glucose (green) superimposed on UDP-a-D-glucuronic acid (gray)