Enzymology

1. "What chemical feature most clearly enables the living cell and organism to function, grow and reproduce? Not the carbohydrate stored as starch in plants or glycogen in animals, nor the deposits of fat. It is not the structural proteins that form muscle, elastic tissue, and the skeletal fabric. Nor is it DNA, the genetic material. Despite its glamour, DNA is simply the construction manual that directs the assembly of the cell's proteins. The DNA itself is lifeless; its language cold and austere. What gives the cell its life and personality are enzymes. They govern all body processes; malfunction of even one enzyme can be fatal. Nothing in nature is so tangible and vital to our lives as enzymes, and yet so poorly understood and appreciated by all but a few scientists.“ Enzymology Arthur Kornberg, 1989 For the Love of Enzymes

2. Enzymology Enzymes cause amazing rate accelerations in chemical reactions, and they do so with absolute fidelity and complete specificity Our goal for this course is to understand how enzymes can accomplish these tasks

3. Conformation final folded arrangement of the polypeptide chain Bonding forces forces that hold the enzyme in its conformation Types of forces electrostatic (charge – charge) hydrogen bonding (XH---:Y) Van der Waals (induce dipolar) Cation- (charge – polar) hydrophobic (nonpolar) Enzyme Structural Terminology

4. Enzyme structure different levels of structural characterization Primary amino acid sequence Secondary backbone arrangement (helix, sheet, bents, coils) Tertiary 3-D arrangement of backbone & side chains Quaternary subunit organization (number and type) Enzyme Structural Terminology

5. Enzyme Structural Terminology Binding site cleft or depression in the enzyme surface that has some affinity and selectivity for binding Substrate binding site site on the enzyme where the reactants bind Catalytic site location where chemical reactions are accelerated Active site substrate binding site plus catalytic site

6. Important Amino AcidsCharged Functional Groups There are only five charged amino acid functional groups

7. Important Amino AcidsPolar Functional Groups There are six additional polar amino acid functional groups

8. The answer is that enzymes will borrow and incorporate additional functional groups as they are needed to catalyze different types of reactions That is the total number of potential functional groups that an enzyme can call on to carry out it’s biological function So, how can enzymes selectively catalyze the thousands of reactions in a living cell with just a few functional groups ?

9. Organic Cofactors A greater diversity of functional groups are required to carry out the wide range of enzyme-catalyzed metabolic reactions

10. Metal Ion Cofactors

11. Enzyme Catalytic Efficiency Which enzyme is the most efficient ? The answer ? – it depends on your criteria ! Radzicka & Wolfenden, Science267, 91 (1995)

12. Transition State Theory of Catalysis

13. Strength of Binding Interactions I. mM to M II. M to nM III. pM to zM II III I Enzyme-transition state binding is in its own category Zhang & Houk, Acc. Chem. Res. 38, 379 (2005)

14. Enzyme-transition state binding • The predicted maximum affinity provided by complementary non-covalent interactions is ~1011 M-1 (G  -15 kcal/mol) • Enzyme-TS affinities average ~ 1016 M-1 Zhang & Houk, Acc. Chem. Res. 38, 379 (2005)

15. Enzyme-transition state binding • What is the source of this extraordinary affinity ? • Covalent interactions, including: • traditional covalent bonds (nucleophilic & electrophilic catalysis) • general acid-base catalysis • proton transfers • low barrier hydrogen bonds Zhang & Houk, Acc. Chem. Res. 38, 379 (2005)

16. Covalent Catalysisacyl group transfer Attack of an enzyme functional group leads to the formation of a covalent acyl-enzyme intermediate The increased stability of this covalent intermediate lowers the transition barrier from substrates to products

17. Covalent Catalysisphosphoryl transfer A similar attack during phosphoryl transfer reactions leads to the formation of a transient tetrahedral intermediate This mode of catalysis can lead to a several-orders of magnitude rate acceleration The amount of catalytic enhancement depends on the barriers for the formation and breakdown of the tetrahedral intermediate and the stability of this intermediate.

18. Acid-base Catalysisimidazole catalyzed hydrolysis 10 to 100-fold rate acceleration

19. Catalysis by Proximityhydrolysis of p-nitrophenyl acetate Rate = 35 M-1 min-1

20. Catalysis by Proximityhydrolysis of p-nitrophenyl acetate How would the rate change if the catalyst was incorporated into the structure of the reactant ? n = 1 rate = 200 min-1 6-fold enhancement n = 2 rate = 840 min-1 24-fold enhancement

21. Catalysis by Strainphosphate ester hydrolysis relative rate = 1 relative rate = 108

22. Transition State Theory of Catalysis A* + B* There is another way to lower the catalytic barrier in addition to stabilizing the transition state Destabilize the substrates !

23. Substrate Destabilization • steric compression • force reactive groups closer together • geometric distortion • put torque on the substrate structure to move it towards the geometry of the product • electrostatic repulsion • introduce similar charges near a charged substrate functional group • desolvation • strip stabilizing solvent interactions

24. Enzyme Specificity The simplest enzyme-catalyzed reaction involves a single substrate being converted to a single product

25. Enzyme Catalysis • enzymes can cause amazing enhancements in the rates of chemical reactions • enzymes have a unique capacity to incorporate multiple modes of catalysis that have a cumulative impact on reaction rates • anything that leads to a lower activation barrier (stabilization of an intermediate, destabilization of the reactants) will result in a rate acceleration • these catalytic accelerations are achieved while maintaining nearly absolute selectivity