Mechanisms of Enzyme Action. Transition (TS) State Intermediate Transition state = unstable high-energy intermediate Rate of rxn depends on the frequency.

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Presentation transcript:

Mechanisms of Enzyme Action

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 secs)

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 TS The higher [TS], the move likely the rxn will proceed. Transition State (TS) Intermediate

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.

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

Common types of enzymatic mechanisms Substitutions rxns Bond cleavage rxns Redox rxns

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

Heterolytic vs homolytic cleavage Carbanion formation (retains both e - ) R 3 -C-H  R 3 -C: - + H + Carbocation formation (lose both e - ) R 3 -C-H  R 3 -C + + H: - Free radical formation (lose single e - ) R 1 -O-O-R 2  R 1 -O* + *O-R 2 Cleavage Rxns Hydride ion

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)

Polar AA Residues in Active Sites ReactiveCharge 7Functions Aspartate-COO - -1Cation Binding, H + transfer Glutamate-COO - -1Cation Binding, H + transfer HistidineImidazole~0H + transfer Cysteine-CH 2 SH~0Binding of acyl groups TyrosinePhenol 0H-Bonding to ligands Lysine-NH Anion binding, H + transfer ArginineGuanidinium+1Anion binding Serine -CH 2 OH 0Binding of acyl groups

Accelerates rxn by catalytic transfer of a proton Involves AA residues that can donate or transfer protons at neutral pH (e.g. histidine) HB + :B + H + 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 1)A-P-P-P(ATP) + E-OH A-P-P (ADP) + E-O-PO 4 - 2)E-O-PO Protein-OH E + Protein-O- PO 4 -

Binding Modes of Enzymatic Catalysis Proximity Effects Transition State Stabilization

Proximity Effects In multi-substrate reactions, collecting and correct positioning of substrates important Increases effective concentration of substrates which favors formation of TS Decreases activation energy by decreasing entropy Increases rate of reaction > 10,000 fold E binding to S can not be too strong or conversion from ES to TS would require too much energy

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

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 Transition state analog Equilibrium between ES TS, enzyme drives equilibrium towards TS Enzyme binds more tightly to TS than substrate

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

Substrate binding specificity