Mechanisms of Enzyme Action Chapter 6 Mechanisms of Enzyme Action

Enzymatic Catalysis Activation Energy (AE) – The energy require to reach transition state from ground state. AE barrier must be exceeded for rxn to proceed. Lower AE barrier, the more stable the transition state (TS) The higher [TS], the move likely the rxn will proceed.

Enzymatic Catalysis S  Ts  P

Transition (TS) State Intermediate Transition state = unstable high-energy intermediate Rate of rxn depends on the frequency at which reactants collide and form the TS Reactants must be in the correct orientation and collide with sufficient energy to form TS Bonds are in the process of being formed and broken in TS Short lived (10–14 to 10-13 secs)

Intermediates Intermediates are stable. In rxns w/ intermediates, 2 TS’s are involved. The slowest step (rate determining) has the highest AE barrier. Formation of intermediate is the slowest step.

Enzyme binding of substrates decrease activation energy by increasing the initial ground state (brings reactants into correct orientation, decrease entropy) Need to stabilize TS to lower activation energy barrier.

ES complex must not be too stable Raising the energy of ES will increase the catalyzed rate This is accomplished by loss of entropy due to formation of ES and destabilization of ES by strain distortion desolvation

Transition State Stabilization Equilibrium between ES <-> TS, enzyme drives equilibrium towards TS Enzyme binds more tightly to TS than substrate Transition state analog

Mechanistic Strategies

Polar AA Residues in Active Sites

Common types of enzymatic mechanisms Substitutions rxns Bond cleavage rxns Redox rxns Acid base catalysis Covalent catalysis

Substitution Rxns Nucleophillic Substitution– Direct Substitution Nucleophillic = e- rich Electrophillic = e- poor transition state

Oxidation reduction (Redox) Rxns Loose e- = oxidation (LEO) Gain e- = reduction (GER) Central to energy production If something oxidized something must be reduced (reducing agent donates e- to oxidizing agent) Oxidations = removal of hydrogen or addition of oxygen or removal of e- In biological systems reducing agent is usually a co-factor (NADH of NADPH)

Cleavage Rxns Heterolytic vs homolytic cleavage Carbanion formation (retains both e-) R3-C-H  R3-C:- + H+ Carbocation formation (lose both e-) R3-C-H  R3-C+ + H:- Free radical formation (lose single e-) R1-O-O-R2  R1-O* + *O-R2 Hydride ion

Acid-Base Catalysis Accelerates rxn by catalytic transfer of a proton Involves AA residues that can accept a proton Can remove proton from –OH, -NH, -CH, or –XH Creates a strong nucleophillic reactant (i.e. X:-)

Acid-Base Catalysis : carbanion intermediate

Covalent Catalysis 20% of all enzymes employ covalent catalysis A-X + B + E <-> BX + E + A A group from a substrate binds covalently to enzyme (A-X + E <-> A + X-E) The intermediate enzyme substrate complex (A-X) then donates the group (X) to a second substrate (B) (B + X-E <-> B-X + E)

Covalent Catalysis Protein Kinases ATP + E + Protein <-> ADP + E + Protein-P A-P-P-P(ATP) + E-OH <-> A-P-P (ADP) + E-O-PO4- E-O-PO4- + Protein-OH <-> E + Protein-O- PO4-

Trypsin, chymotrypsin, elastase, thrombin, subtilisin, plasmin, TPA The Serine Proteases Trypsin, chymotrypsin, elastase, thrombin, subtilisin, plasmin, TPA All involve a serine in catalysis - thus the name Ser is part of a "catalytic triad" of Ser, His, Asp (show over head) Serine proteases are homologous, but locations of the three crucial residues differ somewhat Substrate specificity determined by binding pocket

Serine Proteases are structurally Similar Chymotrpsin Trypsin Elastase

Substrate binding specificity