Lecture 15: Enzymes & Kinetics Mechanisms Margaret A. Daugherty Fall 2004 ROLE OF THE TRANSITION STATE Consider the reaction: H-O-H + Cl - H-O δ- H Cl δ- HO - + H-Cl Reactants Transition state Products In the transition state (denoted by X ), the chemistry is intermediate between reactant and product. The substrates are strained hence chemistry can occur. Enzymes work to reduce G, the barrier between substrate and transition state.
REGULATION AND RECOGNITION LOCK & KEY Lysozyme binding to an antibody INDUCED FIT (enzymes) Hexokinase binding it s substrate glucose BIOC 205 ENZYME-SUBSTRATE INTERACTIONS THE LOCK & KEY MODEL A perfect match between enzyme and substrate can explain enzyme specificity does not explain enzymatic catalysis If everything is such a perfect fit, how does the chemistry occur? How do we go from substrate to product? Implies a rigid, inflexible enzyme
ENZYME-SUBSTRATE INTERACTIONS THE INDUCED FIT MODEL KEY FEATURES: ENZYME STRUCTURE CHANGES IN PRESENCE OF SUBSTRATE --- BRINGS CATALYTIC GROUPS INTO CORRECT POSITION TO DO CHEMISTRY ---SUBSTRATE IS FORCED INTO TRANSITION STATE CONFORMATION ACCOUNTS FOR SPECIFITY AND FOR CATALYSIS Intermediate State in Catalysis uncatalyzed reaction Catalyzed reaction: 2 low barriers replacing 1 high barrier. Lower activation energy; reaction goes faster ES complex with low energy EX complex with lower energy than X 10-13 sec Enzymes stabilize transition state more than ES complex
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 Active Site Small compared to the protein; 3D entity, usually found in a cleft or crevice; Size, shape, charge, non-covalent interactions are all important! Binding is via multiple interactions; Specificity achieved by the arrangement of the active site; Converts substrate to product. Active site
ENZYME ACTIVE SITES BIND THE TRANSITION STATE BETTER THAN SUBSTRATE OR PRODUCT Goal: Perform chemistry! Fast! Binding of Substrate must be favorable;...but not too favorable! Destabilization of ES relative to the transition state, EX Loss of entropy on formation of ES Destabilization of ES by Strain Distortion Desolvation Fig 14.2 Favorable binding energy destabilized by entropy loss, strain, distortion, desolvation! FORMATION OF ES: LOSS OF ENTROPY MAIN POINTS: 2 reactants --> 1 product Lose translational and rotational entropy Entropy offset by favorable H (chemical interactions) No further loss of entropy in going to EX Note: orientation: the active site has the catalytic groups in the most effective orientation to carry out the needed chemistry.
DESTABILIZATION OF ES: Strain, Desolvation, Electrostatic Effects + MAIN POINTS 1). Active site is specialized to bind transition state to carry out chemistry; in order to make a fit the ES complex is strained or destabilized. 2). When charged groups move from solvent to active site, they often become desolvated - this makes them less stable, hence more reactive. 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. Reaction pathway acts to remove stress - hence faster rates result. 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. Hence faster reaction rates.
STUDYING TRANSITION STATE - TRANSITION STATE ANALOGS: High affinity (10-14 M) substrates that mimic the transition state; transition state analogs are stable and only mimic the actual TS. As such they never bind as tightly as the TS itself! AAs Frequently Involved in Catalysis
CATALYTIC FUNCTIONS OF REACTIVE GROUPS OF IONIZABLE AMINO ACIDS O R-C- Acyl group COVALENT CATALYSIS A covalent bond is formed between enzyme and substrate BX + Enzyme E-B + X + Y Enz + BY serine proteases: coming right up!
Acid-Base Catalysis Specific vs. general Specific Acid-Base Catalysis: H + or OH - accelerates the reaction; donated from H 2 0 In this case, the reaction will be ph dependent; 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 - i.e., there is some other group available to act as an general acid or general base. Histidine plays a major role as a general acid or base. pka ~ 7.0
Enolase: Lys - general base; abstracts H+ Glu - general acid; Donating proton to OH --> H 2 0 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 3 - + H + Zinc is tetrahedrally coordinated H 2 O is polarized! Im = imidazole of histidine 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 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 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 Small differences in the nature of the binding pocket give rise to substrate specificity in the proteases. 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. MECHANISM I: BINDING OF SUBSTRATE Asp-102 immobilizes His57 via H-bond His acts as general base; deprotonates Ser; Via nucleophilic reaction, serine attacks C=O Covalent intermediate with protonated amine; negative charge on O is unstable His acts as general acid; donates H to amide nitrogen
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: 1). C-O - bond longer than C=O bond; Amide H s and O closer together 2). O - hydrogen bonds stronger than C=O hydrogen bonds 3). Side chain interactions MECHANISM II: ATTACK OF WATER; RELEASE OF AMINO PRODUCT From last slide: C-N bond cleavage and release of amino product ; resulting acyl intermediate is relatively stable His acts as general base, accepting proton from water; Water attacks (nucleophilic) on carbonyl.
MECHANISM III: COLLAPSE OF TETRAHEDRAL INTERMEDIATE From last slide Transient tetrahedral intermediate; with collapse of tetrahedral intermediate assisted by His donating a proton to Ser (concerted). MECHANISM IV: RELEASE OF CARBOXYL PRODUCT Final step: Deprotonation of carboxyl group; with departure of carboxyl product from active site.
Serine Protease Mechanism http://info.bio.cmu.edu/courses/03231/protease/serpro.htm 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.