Biochemistry Study Guide Chapter 8

Enzymes
The agents of metabolic function
Almost all are proteins some are RNA
Give cells capacity to exert kinetic control over thermodynamic potential a rxn
may have -G but that doesnt dictate how long the rxn takes use an enzyme to
control speed of rxn
Characterized by three distinct features
1. Catalytic Power
2. Specificity
3. Regulation
1. Catalytic Power
Enzymes can accelerate reactions as much as 10 16 over uncatalyzed rates
e.g., urease: catalyzed rate 3x10 4/sec vs uncatalyzed 3x10-10/sec = ratio of
1014
e.g., OMP decarboxylase: rate inhancement ratio = 10 17
Hydration of CO2
is thermodynamically favored but very slow
introduction of carbonic anyhdrase accelerates reaction 10 million-fold!
However it is specific will not hydrate CO, only CO 2
2. Specificity
Several enzymes hydrolyze proteins but these proteolytic enzymes (digestive
enzymes) are quite specific: some proteases will hydrolyze only peptide bonds
containing a set of amino acids with specific R-groups
e.g., trypsin: will only cleave bond if side chain has Lys or Arg
e.g., thrombin: will only cleave peptide bond if side chain has Arg and Gly
Many proteases will also hydrolyze ester bond (similar to peptide bond- has O
instead of NH) hydrolyzation of ester results in acid and alcohol pdts
Enzymes selectively recognize proper substrates over other molecules
Enzymes produce pdts in very high yields, often over 95%
Specificity is controlled by 3D structure of active site: unique fit of substrate
with enzyme controls selectivity for substrate and pdt yield
3. Regulation
Some enzymes require cofactors / prosthetic groups for fxn
Coenzyme small, vitamin derived organic molecules
Metal ion such as Zn, Mg
Apoenzyme: enzyme without its cofactor
Holoenzyme: apoenzyme plus cofactor
Reactions that are not spontaneous can be made spontaneous by adjusting
concentrations of reactants and products
For real rxns, must consider concentrations, not just refer to G
e.g., DHAP isomerization to GAP
under standard state conditions G = +7.5 kJ/mol (will not occur
spontaneously)
if concentrations are altered: GAP = 3x10-6 M and DHAP = 2x10-4 M, the
G = -2.9 kJ/mol (reaction will proceed)

Relation between G and Keq

G = -RTlnKeq
For each 10-fold change in Keq, the G changes by 5.7 kJ/mol
Yield of pure pdt in 10-step rxn: 35% yield in lab, 99.9% in nature because of
enzymatic specificity: no side rxns or by-products occur!
Enzymes do accelerate the reaction rate, but do NOT alter the rxn equil (do not
change G): enzymes lower the free energy of activation by facilitating the
formation of the transition state
Enzymes accelerate rxns by providing a rxn pathway in which the transition state
has a lower free energy and hence is more rapidly formed than in the uncatalyzed
rxn

Transition state (X )
Overall free energy change for rxn (G) related to equilibrium constant (Keq)
Free energy of activation for rxn (G ) related to rate constant (affected by
enzyme)
Transition state is transient molecular structure that is no longer substrate but not
yet pdt
Transition state is least stable and most-seldom-occupied structure along rxn
pathway

Enzyme-Substrate Complex (ES)

Substrate bound to enzyme active site
Evidence for ES complex: influence of substrate concentration on rate of
enzymatic rxn
Maximal velocity: all enzymes
are saturated with substrate
Hyperbolic saturation curve

Active site is 3D cleft or crevice (active site not composed of adjacent residues in
1 sequence, but residues at various locations in sequence that become adjacent
once folded in 3D structure)
ES complex held together by weak noncovalent interactions

Specificity of ES interactions arises mainly from H-bonding and from the shape of
the active site which rejects molecules that dont have a sufficiently
complementary shape
ES complex binding:
Lock & Key: substrate binds active site with complementary shape
Induced Fit: active site assumes shape complementary to substrate only
after S is bound

Chemical kinetics
Reaction rate (velocity) for A P
V = - A / T (decrease of substrate over time) or P / T (increase of pdt
over time)
1st order rxn (dep on one reactant unimolecular rxn)
o A P : V = k[A]
o Rate constant (k) units: s-1
2nd order rxn (dep on 2 reactant molecules bimolecular rxn)
o 2A P : V = k[A]2
o A + B P : V = k[A][B]
o Rate constant (k) units: M-1s-1
Remember Keq = k1 / k-1 where k1 is forward rate constant, k-1 is reverse rate
constant
Time-dependent concentration changes of [S], [ES], [P], [E]
(remember enzyme is catalyst and cannot be lost or changed during rxn)

Reach ES complex rapidly!

Michaelis-Menten Equation
Rate of formation of product when an enzyme (E) combines with a substrate (S) to
form an enzyme-substrate complex (ES) which can proceed to form product (P) or
dissociate into E and S
Assumes formation of ES complex
Assumes that ES complex is in rapid equilibrium with free enzyme

Assume that breakdown of ES to form pdts is slower than formation of ES and

breakdown of ES to reform E and S: E + S

ES

E+P

k1 forward
k-1 reverse

k2

M-M Equation: V0 = Vmax[S] / Km + [S]

V0 is initial velocity (when t=0)
Vmax is maximal velocity
reaction rate when enzyme is fully saturated with substrate
is theoretical in reality we never reach Vmax
equal to the pdt of k2 and the total concentration of enzyme
Km (Michaelis constant) is the substrate concentration at which the reaction rate is
half maximal

When Km = [S], V0 = Vmax

Vmax = maximal velocity

= Michaelis constant

Michaelis constant (Km)

Substrate conc at which rxn rate is maximal
Km is derived from rate constants: Km = (k-1 + k2) / k1
Km is an estimate of the dissociation constant of E from ES
Small Km means tight binding (less dissociation)
High Km means weak binding (more dissociation)

E+S

Theoretical maximum velocity (Vmax)

Vmax is a constant
Vmax is never achieved in reality: would require that all enzyme molecules tightly
bound with substrate
Vmax is asymptotically approached as substrate is increased
Double-reciprocal (Lineweaver-Burk) plot
Old method to derive Km and Vmax now use computer program to determine
Dual

nature of Michaelis-Menten equation

Generated
by plotting 1/V0 over
Combination of 0-order and 1st-order
kinetics
1/[S]
When S is low, equation for rate
is 1st order in S { V0 = (Vmax/Km)[S] }
Slope
is Km / Vinmax
When S is high, equation for rate
is 0-order
S { V 0 = Vmax }
Intercept
axis hyperbolic
is
The Michaelis-Menten equation describeson
a vertical
rectangular
dependence of
1/V
max
V on S
Intercept on horizontal axis is
1st-order
1/Km 0-order

Allosteric enzymatic rxns do not follow M-M kinetics

Allosteric enzymes consist of multiple subunits
What weand multiple active sites
actually binding
Sigmoidal curve (not hyperbolic) cooperative
observe

Variation of Km values of enzymes

Chymotrypsin Km = 5000 M
Lysozyme Km = 6
Variation in Km also depends on what substrate enzyme is acting upon:
Pyruvate carboxylase on pyruvate Km = 400
on HCO3 Km = 1000
on ATP Km = 60
Enzyme Mechanisms
1. Turnover number
2. Catalytic efficiency
3. Enzyme inhibitors
4. Covalent Catalysis
5. General Acid-Base Catalysis
1. Turnover number
Measure of catalytic activity (minimum catalytic activity = k cat)
kcat is the number of substrate molecules converted to pdt per enzyme
molecule per unit of time, when enzyme is saturated with substrate
Large variation: kcat values range from less than 1 per second to many millions
per second
If M-M model fits: kcat = k2 = Vmax/Etotal
2. Catalytic Efficiency
Name for kcat / Km
2nd-order rate constant
Measure of how the enzyme performs when substrate concentration is low
Upper limit for catalytic efficiency is the diffusion limit the rate at which E and
S diffuse together (between 108 and 109 M-1s-1) because in perfect scenario E
and S diffuse, find each other, and form ES complex limited only by rate of
diffusion
So almost perfect catalytic efficiency is 108 to 109
Varies based on substrate e.g., chymotrypsin catalytic efficiency highest when
substrate has bulky, hydrophobic side chain such as Phe (k cat / Km = 105) vs
simple side chain such as Gly (kcat / Km = 10-1)
Multiple substrate reactions A + B P + Q
Ordered sequence rxns: must bind & release in sequential order (bind A, bind B,
release P, release Q)
Random sequential rxns: either substrate can bind first and/or either pdt can be
released first
Double-displacement (ping pong) rxns: (bind A, release P, bind B, release Q)

3. Enzyme Inhibitors
Reversible inhibitors interact with an enzyme via noncovalent associations
rapid equilibrium between enzyme and inhibitor

Irreversible inhibitors interact with an enzyme via covalent associations

inhibitor is covalently linked to enzyme or bound so tightly that its dissociation
from enzyme is very slow (provides ability to map 3D structure of active site)
Classes of inhibition:
Competitive inhibition inhibitor binds only to E, not to ES (binds at
substrate binding site)
o Prevents substrate from binding to active site
o Reduces rxn velocity by decreasing proportion of enzyme molecules
bound to substrate
o Can be overcome by raising substrate concentration
Noncompetitive inhibition inhibitor can bind either E or ES (but I and S bind
to E at diff sites)
o Inhibitor decreases turnover number
o Cannot be overcome by raising substrate concentration
Uncompetitive inhibition inhibitor binds only to ES, not to E alone
o Cannot be overcome by raising substrate concentration

Competitive Inhibition
If you increase [S] you can
knock inhibitor off enzyme
* Vmax is same but Km is
increased *

-1/KM 1/Vmax

-1/KM(1+[I]/Ki)
Noncompetitive inhibition
* Vmax decreases *
* Km is unchanged *

Uncompetitive inhibition
* Both Vmax and Km are
lowered *

Irreversible inhibitors
Group-specific reagents reagents react with specific side chains of amino acids
e.g., Acetycholinesterase reaction with DIPF (specifies Ser-OH)
Affinity labeling reactive substrate analogs structurally similar to substrate for
enzyme and covalently bind to active-site residues; are more specific for enzyme
active site than group-specific
e.g., Chymotrypsin substrate and inhibitor TPCK similar structure
Suicide inhibitors (mechanism-based inhibitors) modified substrates that provide
most specific means to modify an enzyme active site; inhibitor binds to enzyme as
a substrate and initially processed by the normal catalytic mechanism which then
generates a reactive intermediate that inactivates the enzyme through covalent
modification
e.g., N,N-dimethypropargylamine and (-)Deprenyl inhibition of monoamine oxidase
(MAO) MAO deaminates NTMs (such as dopamine in the case of Parkinsons
disease), inhibition drugs
Transition-state analogs (mimics) potent enzyme inhibitors that mimic the
transition state structure an enzyme binds the transition state more tightly than
the substrate
e.g., Proline isomerization catalyzed by proline racemase enzyme: proline enters
trigonal planar transition state mimicked by pyrrole 2-carboxylic acid (inhibitor)
that binds racemase 160 times stronger than proline

Penicillin
Irreversibly inactivates a key enzyme in bacterial cell wall synthesis
Contains reactive peptide bond in -lactam ring that reacts with enzyme
Cell wall structure: polysaccharide chains are cross-linked with pentaglycine and
tetrapeptide: formation of which is achieved via enzyme Glyopeptide
transpeptidase
Penicillin blocks the last step in cell wall synthesis (cross-linking of different
peptidoglycan strands) by mimicking the terminal D-Ala-D-Ala unit of normal
substrate
Most enzymes are proteins, but
Ribozymes segments of RNA that display enzyme activity in absence of protein
e.g., RNaseP and peptidyl transferase
Abzymes antibodies that recognize the transition state, bind, and catalyze rxn