Enzyme function: the transition state. Enzymes & Kinetics V: Mechanisms. Catalytic Reactions. Margaret A. Daugherty A B. Lecture 16: Fall 2003

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1 Lecture 16: Enzymes & Kinetics V: Mechanisms Margaret A. Daugherty Fall 2003 Enzyme function: the transition state Catalytic Reactions A B Catalysts (e.g. enzymes) act by lowering the transition state free energy for the reaction being catalyzed.

2 Intermediate State in Catalysis Uncatalyzed reaction Catalyzed reaction sec LARGE RATE ACCELERATIONS: ENZYME STRUCTURE AND MECHANISM MECHANISMS OF CATALYSIS 1). Entropy loss in the formation of ES 2). Destabilization of ES due to strain, desolvation or electrostatic effects 3). Proximity and orientation 4). Covalent catalysis (an example of the serine proteases) 5). Metal ion catalysis 6). General acid or base catalysis Any or all of this things can contribute to catalytic rate acceleration

3 FORMATION OF ES: LOSS OF ENTROPY MAIN POINTS: Entropically unfavorable Enthalpically favorable No unfavorable entropy in ES --> EX Note: orientation of active site is optimal for chemistry DESTABILIZATION OF ES: Strain, Desolvation, Electrostatic Effects + MAIN POINTS 1). Active site is specialized to bind transition state to carry out chemistry 2). When charged groups move from solvent to active site, they often become desolvated.

4 DESTABILIZATION OF ES: Strain, Desolvation, Electrostatic Effects MAIN POINT Charged groups on S may be forced to interact with like charges. This is unfavorable and destabilizes S. Note: proximity Bringing the substrate together with the catalytic groups on the enzyme results in an effective concentration increase relative to the concentration of substrates in solution. STUDYING TRANSITION STATE ANALOGS: HIGH AFFINITY (10-14 M) SUBSTRATESTHAT MIMIC THAT THE TRANSITION STATE

5 AAs Frequently Involved in Catalysis CATALYTIC FUNCTIONS OF REACTIVE GROUPS OF IONIZABLE AMINO ACIDS O R-C- Acyl group

6 Acid-Base Catalysis Specific Acid-Base Catalysis: H + or OH - accelerates the reaction; donated from H 2 0 However buffers that can donate or accept H + /OH - will not affect the reaction rate: Acid-Base Catalysis General Acid-Base Catalysis: in which an acid or base other than H + or OH - (other than H 2 O) accelerates the reaction; reactive groups in the enzymes active sites; These are characterized by changes in rate with increasing buffer concentrations

7 GENERAL ACID/BASE CATALYSIS UNCATALYZED GENERAL ACID GENERAL BASE Enolase:

8 Metal Ion Catalysis Metalloenzymes: bind metal tightly require metal for 3-D structure Transition metal ions Fe 2+, Fe 3+, Zn 2+, Mn 2+ or Co 2+ Metal activated enzymes: bind metals weakly; usually only during catalysis - play a structural role; bind metals from solution alkali and alkaline earth metals Na +, K +, Mg ++ or Ca ++ Roles: Bind to substrates and orient the substrates Mediate redox reactions through reversible changes in the metals oxidation state Electrostatically shield or stabilize negative charges. Human Carbonic Anhydrase: A Zinc containing enzyme CO 2 + H 2 O <---> HCO H + Zinc is tetrahedrally coordinated H 2 O is polarized!

9 THE SERINE PROTEASES Trypsin, chymotrypsin, elastase, thrombin, subtilisin, plasmin, TPA All involve serine in their catalytic mechanism; Serine is part of a catalytic triad of Ser, His, Asp All serine proteases are homologous, but locations of the three critical residues vary. By convention, numbering of critical residues is always the same: His-57, Asp-102 and Ser-195 PRIMARY STRUCTURE OF SERINE PROTEASES

10 ZYMOGENS ARE CLEAVED TO THEIR ACTIVE CONFORMATION ACTIVE SITE: A DEPRESSION ON PROTEIN SURFACE Chymotrypsin in complex with eglin C depth of active site depression depends on reaction

11 Chymotrypsin, trypsin and elastase blue yellow green All three proteases show: similar backbone conformations active site residue orientations yet. All three exhibit different cleavage specificity Chymotrpysin: aromatics; trypsin: basic & elastase: gly & alanine SUBSTRATE SPECIFICITY shallow hydrophobic deep hydrophobic deep negatively charged

12 TRYPSIN - TRYPSIN INHIBITOR COMPLEX

13 ARTIFICIAL SUBSTRATES PROVIDE INSIGHT INTO MECHANISM EVENTS AT THE ACTIVE SITE: MECHANISM COVALENT & GENERAL ACID-BASE CHEMISTRY 1). Asp-102 functions to orient His-57 2). His-57 acts as a general acid and a general base. 3). Ser-195 covalently binds the peptide to be cleaved 4). Covalent bond formation turns a trigonal C into a tetrahedral carbon 5). A tetrahedral oxyanion intermediate is stabilized by NH s of Gly193 and Ser195.

14 MECHANISM I: BINDING OF SUBSTRATE Oxyanion Hole Major Role in Transition State Stabilization Recall: binding site is relatively hydrophobic; need to neutralize charges Substrate C=0 hydrogen bonds to amide Hs of G193 and S195 Enhanced stabilization in the transition state

15 MECHANISM II: ATTACK OF WATER; RELEASE OF AMINO PRODUCT MECHANISM III: COLLAPSE OF TETRAHEDRAL INTERMEDIATE From last slide

16 MECHANISM IV: RELEASE OF CARBOXYL PRODUCT Review 1). Enzymes stabilize transition state more than ES complex. 2). There are a limited number of factors that contribute to catalysis. 3). Destabilization of ES relative to the transition state occurs via: Loss of entropy on forming ES Strain on the ES complex Distortion of the ES complex Desolvation of the ES complex 4). Catalysis is also achieved through orientation of the chemically reactive groups on the enzyme in close proximity with the substrate. 5). Transition state analogs are a way of studying enzyme mechanism. 6). The serine proteases have a catalytic triad that consists of Ser, His and Asp. The job of Asp102 is to correctly position His57 in the active site. His57 acts both as a general acid and a general base. Serine covalently binds the substrate. Water is involved in the mechanism.