Chapter 11 Enzymatic Catalysis & Activation energy and the reaction coordinate, Catalytic mechanisms

Enzymes = catalytically active biological macromolecules; powerful biological catalysts

  • most are globular proteins, some RNA (like ribozymes & ribosomal RNA) molecules also catalyze reactions

  • by catalyzing a reaction, enzymes help ensure

    • higher reaction rates (both forward AND reverse reaction increased)

      • accelerating chemical reactions

      • mediated reactions often be several orders of magnitude faster than the unmediated chemical transformation

    • greater reaction specificity - enzymes can make the desired reaction the most favorable

      • act on specific substrates but many enzymes also react with broad classes of molecules

      • specificity is determined by steric (space & shape) considerations

      • specific binding is necessary but not sufficient for enzymatic activity

            sterospecific enzyme-substrate interactions

  •         they also afford speciifc products

    •     product sterochemisty is highly controlled in enzymes including chirality

      •        citrate: prochiral            isocitrate: only one enantiomer produced

      • nearly all enzymes that produce chiral products are absolutely stereospecific

  • enzymes can promote reaction specificity

  • some enzymes are more permissive 

    • non-specific enzymes

      • work on a # of substrates

      • alcohol dehdrogenase will oxidize methanol, ethanol, propanol

      • chymotrypsin will hydrolyze amides as well as esters

  • by catalyzing a reaction, enzymes help ensure

    • milder reaction conditions (temps below 100 °C, atmospheric pressure, nearly neutral pH)

    • capacity for regulation (allosteric control, covalent modification of enzymes, variation of amounts of enzymes synthesized)

enzyme classification (accepted & systematic name)

  • named after the reactions they catalyze

  • “ase” suffix - added to either substrate or reaction name

  • 6 major classes of systematic names

  1. oxidoreductases:     oxidation-reduction reactions

  2. transferases:           transfer of functional groups

  3. hydrolases:              hydrolysis reactions

  4. lyases:                     group elimination to form double bonds

  5. isomerases:             isomerization

  6. ligases:                     bond formation coupled with ATP hydrolysis

oxidoreductases - enzyme that catalyzes the transfer of electrons from one molecule to another molecule. NADP & NAD+ often are cofactors in oxireductase catzlyzed reaction

  • A- + B → A + B-        A- = electron donor (reductant)       B- = electron acceptor (oxidant)

  • ex: glyceraldehyde-3-phosphate → (glyceraldehyde phosphate dehydrogenase) →     D-glycerate 1,3-biphosphate

transferase - enzyme that catalyzes the transfer of a functional group from one molecule to another molecule

  • A-X + B → A + B-X        X = methyl or phosphate group

hydrolase - enzyme that catalyzes the hydrolysis of a chemical bond

  • A-B + H2O → A-OH + B-H

lyase - enzyme that catalyzes the breaking of various chemical bonds by means other than hydrolysis and oxidation, forming new double bond or new ring structure

  • cleavage of C-C, C-O, C-N, etc

isomerase - enzyme that catalyzes the structural rearrangement of isomers

ligase - enzyme that catalyzes the joining of 2 large molecules by forming a new chemical bond. enzymes which catalyze joining of C-O, C-S, C-N etc

some enzymes require cofactors

  • enzymes mediate chemical reactions through their amino acid side chain functional groups

    • (ex: amylase uses aspartate & glutamate side chain carboxylic acid)

  • however a non-amino acid unit is required to carry out the chemistry (aka cofactors)

  • cofactors expand the range of enzymatic reactions

  • can be transition metal ions, metal containing molecules, or organic compounds

  • cofactors → metal ions or coenzymes→ co substrates or prosthetic groups

cofactor types

  • single/clusters/metal ions

    • Fe2+, Mn2+, Cu2+, Zn2+

  • coenzymes

    • permanent or transitory organic/inorganic molecules that can carry out key functions

      • co-subtrates: transiently associated

      • prosthetic groups: permanently associated with protein

  • apoproteins/apoenzymes = proteins that lack cofactors 

  • holoproteins/holoenzymes = proteins with their cofactors

    • apoenzyme (inactive) + cofactor → holoenzyme (active)

  • example cofactor with metal containing prosthetic group - ctochrome P450 & Nitrogenase

  • example cofactor of metal ion: carbonic anhydrase is enzyme that requires a metal ion cofactor

NAD(P)+

adenosine, D-ribose, nicotinamide (oxidized form) + 2 H+ → reduced form of nicotinamide

properties of enzymes summary

  • act as catalyst but differ from ordinary chemical catalysts in that enzyme-catalyzed reactions exhibit

    • higher reaction rates

    • milder reaction conditions

    • greater reaction specificity

    • capacity for regulation

  • enzymes act on specific substrates

  • some enzymes require co-factors

  • enzymes increase reaction rates by lowering the activation energy (enzyme = catalyst) but dont affect equilirbirum constants

PART II Activation energy and the reaction coordinate & Catalytic mechanisms

a bimolecular reaction A + B-C → A-B + C at some point in the reaction coordinate, an intermediate ternary complex will exist:     A…B…C 

transition state = process of bond formation and bond breakage

Ex: Ha + Hb-Hc → Ha-Hb + Hc        Ha…Hb…Hc

reaction coordinate diagrams

  • describes the thermodynamics and path dependence of chemical reaction 

  • thermodynamics do not change (relative energies of starting & ending points)

    • state functions

  • course of the reaction can change and moves through at least 1 transition state

    • transition states cannot be isolated

  • energy changes include the activation energy (height of transition state, Ea or delta G*)

reaction rate is proportional to e^(G/RT) 

  •     the greater the value of delta G the slower the reaction rate

multi-step reactions have rate determining steps 

   k1   k2

A → I → P

  • if one reaction step is much slower than the rest the step acts as a ‘bottleneck’ and is rate-limiting step

  • transition state, since this the point of highest energy fr that step in the reaction, the difference between reactant and transition state freee energy is known as the activation barrier

  • for each of the reactions on the left there are 2 transtion states and 1 intermediate

catalysis lowers activation energy

  • being catalzed by enzyme

  • lowering Ea energies makes rate constants larger = reaction s fater

  • reduction in Ea afforded by an enzyme is known as delta delta G ++ which = delta G ++ (uncat) - delta G ++ (cat)

  • the presence of catalyst does not affect the overal delta G of reaction but lowers the Ea

  • energy barrier/activtion energy is lowered by the same amount for both the forward and reverse reaction

rate of reaction is increased by e^(delta delta G++ (cat)/RT)

  • delta delta G ++(cat) = 5.71 kJ/mol is 10 fold increase in rate (half of H bond)

  • delta delta G ++(cat) = 34.25 kj/mol produces a million fold increase in rate

  • rate enhancement is a sensitive function of delta delta G ++ cat

enzyme-catalyzed reactions take place within the active site

active site = provides a specific environment in whihc a given reaction can occur more rapidly

substrate = the molecule that is bound to the active site and acted upon by the enzyme

enzyme affect reactiion rates not equilibria (enzymes are not used up in the process)

simple enzymatic reactions: E + S = ES = EP = E + P

E = enzyme     S = substrate    P = product

ES & EP = transient complexes of enzyme

enzyme functions - lower Ea & increase rate of reaction but doesn’t change equilibrium

  1. perferential binding of the transition state complex: enzymes provide binding energy that conteracts the Ea (form of energy couple)

    1. enzyme can bind to substrate (* but doesnt want enzyme to bind to substrate too well)

  2. enzymes or enzymes cofactors can provide functional grouos that allow for an alternative lower E reaction path

    1. acid base catalysis

    2. covalent modification

    3. metal ion based catalysis

  3. proximity and orientation effects: enzymes cna orient the functional groups within the substrate that need to react with each other

  1. perferential binding of the transition state complex: enzymes provide binding energy that conteracts the Ea (form of energy couple)

Scenario 1: enzyme binds substrate very vell (lock key mech)

a) no enzyme: substrate (metal stick) → transition state (bent stick) - high Ea → products (broken stick)

b) enzyme complementary to substrate: magnets - no energy to drive to break ES part - stable complex

  1. decrease in entropy as bring enzyme and substrate together

  2. binding energy provided by interactions between enzyme and substrate 

Scenario 2: induced fit mech allows enzyme to bind to transition state even better than to original substrates

c) enzyme complementary to transition state: ES → ++ → E + P

  • involves conformational change of protein

binding energy compensates for many thermodynamically unfavorable things that have to happen in order for the reaction to occur

  • binding energy is result of multiple noncovalent interactions between

  1. enzyme & substrate AND

  2. enzyme & transition state

  • binding energy compensates for

  1. entropy reduction

  2. free energy required to break the stick/make the reaction happen (catalysis)

vatalysis via perferential transition state binding

  • in addition to stablizing binding of reactants/products of a reaction an enzye can also stabilize the structure of a transition state

  • by having an active site that preferentially binds a transition state, the barrier to forming the species is appreciably lowered

  1. enzymes or enzymes cofactors can provide functional grouos that allow for an alternative lower E reaction path

  1. acid base catalysis occurs by proton transfer

general acid catalysis = proton transfer from an acid loweres the free energy of a reaction’s transition state

general base catalysis = rate is increased by proton abstraction by a base

  • enzymes can provide functional groups that act as an acid catalyst or base catalyst

general acid-base catalysis = proton transfer mediated by molecule other than water

ex: proton donating or proton accepting amino acids

concerted acid base catalysis (ex RNAse) = ability of enzymes to arrange several catalytic groups around their substrates make concerted acid-base catalysis a common mechanism

enzymes can provide functional groups that help promote a certain reaction mechanism…

  1. covalent catalysis = can promote formation of transient covalent bonds between enzyme and substrate altering reaction pathway

     H2O                                                  H2O

A-B → A + B    vs    A-B + X: → A-X + B → A + X: + B

  • covalent catalysis = accelerates reactions rates through transient formation of catalyst-substrate covalent bond

    • common mech to form covalent bond is nucleophilic attack

      • nucleophilic group on catalyst reacts with electrophilic group on substrate

      • alters reaction pathway allowing for different transition state intermediate

aceotactate → CO2 and enolate + H+ → acetone

        v (RNH2 → OH-)                                    v (OH- → RNH2)

[ schiff base (imine) → CO2 and resonance + H+ → resonance ]

3 stages of covalent catalysis

  1. nucleophilic reaction between catalyst and substrate to form covalent bond

  2. withdrawl of e- from reachion center by now electrophilic catalyst

  3. elimination of catalyst, reaction that is essentially reverse of stage 1

       

  1. metal ion cofactors can…

  • help orient substrate molecules or stablize protein structure but is less of catalytic activity & more of a general protein folding/stability activity

  • help stabilize charged reaction transition states (electrostatic catalysis)

  • can participate in redox reactions (metals can reversibly change their oxidation state)

  • positiviely charged metals can draw e- towards them, thereby faciltating downstream reaction mechanims (or allows molecules to more easily release a proton and in situations like this metal acts as a pKa shifter)

  • [ nearly 1/3 of all known enzymes require 1 or more metal ions for catalytic activity ]

role of Zn2+ in carbonic anhydrase

  • OH- comes from H2O that gave up its proton

  • zinc ion makes it bound water molecule so acidic (pKa shifter) that the water gives ups proton)

  1. proximity and orientation effects: enzymes cna orient the functional groups within the substrate that need to react with each other

enzymes can orient the reacting functional groups

proximity & orientation effects = reactants must come together with the proper spetial relationship for a reaction to occur

  • proximity - reacting chemical gorups held near each other

  • orientation effects - chemical groups aligned in proper orientation for reaction to occur (ex orbitals)

  • freeze reacting groups - limit movements of reacting groups

summary of catalytic mechanisms

  • enzymes can be broadly categorized as enhancing that rates of chemical reactions throguh 5 types of mechanisms

    • perferential binding of transition state complex

    • acid-base catalysis

    • covalent catalysis

    • metal ion catalysis

    • proximity & orientation effects

  • the concepts & mechs observed in enzymatic systems are broadly applicable to any type of catalyst