Dignam – Enzymes
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Reaction rate is accelerated; equilibrium distribution (and thus, equilibrium constant) of
reactants and products is not changed
types
o oxidoreductases
o tranferases (e.g. synthases)
o hydrolases
o lyases
o isomerases
o ligases (e.g. synthetases)
Transition state
o if increase concentration of TS, will increase rate of reaction
Equilibrium is reached much more rapidly in the presence of an enzyme, but the
equilibrium constant is not changed
entropy – the ability of a system to occupy many different states
o may use energy to constrain this number of states  a decrease in entropy
o a reaction that results in a decrease in enthalpy and an increase in entropy is a
favorable one
ABC (acid-base catalysis)
o in a non-polar environment, acidic and basic residues exhibit increased activity
via altered pKa
Nucleophilic (covalent) catalysis
o electron pair of nucleophile attacks electron deficient substrate
o e.g. DNA topisomerase I
Active site is often hydrophobic (lower dielectric constant  stronger electrostatic
interactions)
Ribonuclease
o residues in the active site
 general acid/base: H119, H12
 stabilization of negative charge on the phosphate: K41
Aspartyl proteases (use of ABC)
o e.g. pepsin, HIV protease, renin
o involve pairs of carboxyl groups on aspartyl side chains to cleave peptide bonds
 the first residue acts as a base and abstracts a proton, while the second
acts as an acid and protonates the nitrogen
Serine proteases
o e.g….
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o chymotrypsin
 bulky and hydrophobic residues
 acyl intermediate
 catalytically active residues : S195, H57, D102
 S195,H57 = acid/base catalysis
 D102 = uses H bond to orient H57 with S195
o S195 forms ester with substrate (nucleophilic catalysis)
 catalysis is a series of steps
 bind substrate, form tet. intermediate, release, add water
(hydrolyses E-S bond) and reform catalytic portions, form second
tet. intermediate, release, reform original catalytic portions
o trypsin
 positively charged residues
o elastase
 small and aliphatic residues
Michaelis-Menten Equation
o when [S] is small, reaction is almost 1st order
o when [S] is large, v = vmax
Dignam – Enzymes cont’d
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Briggs-Haldane Steady-State Approximation
o more general
o Km = (kcat + k-1)/k1
Double Reciprocal Equation
o Y = 1/v
o m = Km/Vmax
o X = 1/[S]
o b = 1/[Vmax]
Enzyme inhibitors
o Competitive
 same site bound by inhibitor and substrate
 effect may be reversed by increasing [substrate]
 apparent Km for substrate is increased
 velocity of reaction doesn’t change with increasing [inhibitor]
 double reciprocal plot shows same y-intercept, but greater slope
o Noncompetitive
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 velocity decreases with increasing [inhibitor]
 double reciprocal plot shows same x-intercept, but greater slope
o Multi-reactant systems
 sequential
 ping-pong
o “Km” and “Turnover number” slides to be on blackboard
o Irreversible inhibitors
 covalent bond with some part of enzyme  inactivation of enzyme
 interferes with an AA involved in S binding or catalysis
o Suicide inhibitors
 substrates that are converted (by the enzyme) to irreversible inhibitors
 e.g. allopurinol  xanthine oxidase (for gout treatment)
Enzyme regulation
 [S], allosteric effectors (+ or -), covalent mods (+ or -), [E] (mRNA)
Allosterism and cooperativity
o allosteric effectors may affect Vmax and Km
 homotropic, heterotropic
Enzymes and disease
o mutations  inactivity, instability, defective mRNA, promoter inactivation
o deficient activity
ACE inhibitors
CPK isozymes
o CPKMB levels rise following an MI, but not after other events (post-cath., abd.
surg., etc.)
Dignam – Myoglobin, Hemoglobin, and Enzymes
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Myoglobin
o Heme – held in hydrophobic pocket
 Fe-protoporphyrin IX
 heme binds iron when it is in its ferrous (Fe2+) oxidation state
 oxygen binding site
o F8-His, E7-His
o O2 saturation
 dissociation constant = Kd = concentration at which 50% saturation is
achieved
Hemoglobin
o effector sites: protons, 2,3-BPG, CO2, O2
o O2 saturation plot – know how the curve shifts
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Hill Equation – ligand binding to multiple sites
o indicates extent of cooperative binding, and if the binding is positive or negative
o know what the curve should look like if no cooperativity (n = 1), positive
cooperativity (n > 1), or negative cooperativity (n < 1)
o larger K of Hb allows the protein to respond to the need for oxygen (as opposed
to the smaller K for Mb)
Monod model for positive cooperativity of O2 binding to Hb
o R state – relaxed; high affinity
o T state – taut; low affinity