Transcript Enzyme Kinetics

ENZYMES
KINETICS, INHIBITION, REGULATION
Muhammad Jawad Hassan
Assistant Professor
Biochemistry
Michaelis-Menten kinetics
V0 varies with [S]
Vmax
approached
asymptotically
V0 is moles of product
formed per sec. when [P]
is low (close to zero time)
E + SESE + P
Michaelis-Menten Model
V0 = Vmax x[S]/([S] + Km)
Michaelis-Menten Equation
Steady-state & pre-steady-state conditions
At equilibrium,
no net change of [S] & [P]
or of [ES] & [E]
At pre-steady-state,
[P] is low (close to zero
time), hence, V0 for
initial reaction velocity
At pre-steady state, we can ignore the back reactions
Michaelis-Menten kinetics (summary)
Enzyme kinetics (Michaelis-Menten Graph) :
At fixed concentration of enzyme, V0 is almost linearly proportional
to [S] when [S] is small, but is nearly independent of [S] when [S]
is large
k1
k2
Proposed Model: E + S  ES  E + P
ES complex is a necessary intermediate
Objective: find an expression that relates rate of
catalysis to the concentrations of S & E, and the rates
of individual steps
Michaelis-Menten kinetics (summary)
Start with: V0 = k2[ES], and derive,
V0 = Vmax x[S]/([S] + Km)
At low [S] ([S] < Km), V0 = (Vmax/Km)[S]
At high [S] ([S] > Km), V0 = Vmax
When [S] = Km, V0 = Vmax/2.
Thus, Km = substrate concentration at which
the reaction rate (V0) is half max.
Range of Km values
Km provides approximation of [S] in vivo for many enzymes
Lineweaver-Burk plot (double-reciprocal)
Allosteric enzyme kinetics
Sigmoidal dependence of V0 on [S], not Michaelis-Menten
Enzymes have multiple subunits
and multiple active sites
Substrate binding may be cooperative
Enzyme inhibition
A competitive inhibitor
Methotrexate
A competitive inhibitor of dihydrofolate reductase - role in purine
& pyrimidine biosynthesis
Used to treat cancer
Kinetics of competitive inhibitor
Ki =
dissociation
constant for
inhibitor
Increase [S] to
overcome
inhibition
Vmax attainable,
Km is increased
Competitive inhibitor
Vmax unaltered, Km increased
Kinetics of non-competitive inhibitor
Increasing [S] cannot
overcome inhibition
Less E available,
Vmax is lower,
Km remains the same
for available E
Noncompetitive inhibitor
Km unaltered, Vmax decreased
Enzyme inhibition by DIPF
Group - specific reagents react with R groups of amino acids
diisopropylphosphofluoridate
DIPF (nerve gas) reacts with Ser in acetylcholinesterase
Affinity inhibitor: covalent modification
Catalytic strategies commonly employed
1. Covalent catalysis.
The active site contains a
reactive group,
usually a nucleophile that becomes
temporarily covalently modified in the course of
catalysis
2. General acid-base catalysis. A chemical reaction is
catalyzed by an acid or a base. The acid is often the
proton and the base is often a hydroxyl ion. A molecule
other than H2O may play the role of a proton donor or
acceptor.
3. Metal ion catalysis. Metal ion can function in
several ways;
• can serve as an electrophile, stabilizing a
negative charge on a reaction intermediate.
• can generate a nucleophile by increasing the
acidity of a nearby molecule, such as H2O in the
hydration of CO2 by carbonic anhydrase.
• can bind to substrate, increasing the number
of interactions with the enzyme.
4. Catalysis by approximation. Bringing two substrates
together along a single binding surface on an enzyme
Enzyme specificity: chymotrypsin
Cleaves proteins on carboxyl side of aromatic, or large
hydrophobic amino acid
Bonds cleaved, indicated in red
The enzyme needs to generate a powerful nucleophile to cleave the bond
A highly reactive serine (#195) in chymotrypsin
27 other serines not reactive to DIPF,
Ser 195 is a powerful nucleophile
DIPF: di-isopropylphosphofluoridate, only reacts with Ser 195
Covalent catalysis
Hydrolysis in two stages
Acylation to form
acyl-enzyme intermediate
Ser 195 OH group
attacks the carbonyl group
Deacylation to regenerate
free enzyme
Acyl-enzyme intermediate
is hydrolysed
Chymotrypsin in 3D
3 chains; orange,
blue, & green
Catalytic triad of
residues, including
Ser 195
2 interstrand, &
2 intrastrand
disulfide bonds
See Structural Insights
Synthesized as chymotrypsinogen
Proteolytic cleavage to 3 chains
The catalytic triad (constellation of residues)
Ser 195 converted into a potent nucleophile, an alkoxide ion
Asp 102
orients
His 57
Imidazole N as
base catalyst,
accepts H ion,
positions &
polarizes Ser
H ion withdrawal from
Ser 195 generates
alkoxide ion
Regulatory Strategies: Enzymes & Hemoglobin
1. Allosteric control. Proteins contain distinct regulatory sites and
multiple functional sites. Binding of regulatory molecules triggers
conformational changes that affect the active sites.
Display cooperativity: small [S] changes - major activity changes.
Information transducers: signal changes activity or information
shared by sites
2. Multiple forms of enzymes (isozymes). Used at distinct locations or
times. Differ slightly in structure, in Km & Vmax values, and in
regulatory properties
3. Reversible covalent modification. Activities altered by covalent
attachment of modifying group, mostly a phosphoryl group
4. Protleolytic activation. Irreversible conversion of an inactive form
(zymogen) to an active enzyme
Aspartate transcarbamoylase reaction
Committed step in pyrimidine synthesis: inhibited by end product
CTP
CTP inhibits ATCase
CTP stabilizes the T state
CTP binds to
regulatory
subunits
R and T states in equilibrium
ATCase displays sigmoidal kinetics
Substrate binding to one active site converts enzyme to R state
increasing their activity: active sites show cooperativity
Basis of sigmoidal curve
R & T states equivalent to 2 enzymes with different Kms
Cooperativity
Effect of CTP on ATCase kinetics
CTP stabilizes the T state, curve shifts to right
Effect of ATP on ATCase kinetics
ATP, allosteric activator, stabilizes R state, curve shifts to left
Oxygen delivery by hemoglobin, cooperativity enhanced
98 - 32 = 66%
63 - 25 = 38%
Cooperativity
enhances
delivery 1.7 fold
Partial pressure of oxygen
Heme group structure
4 linked pyrrole rings
form a tetrapyrrole
ring with a central
iron atom.
side chains attached
Position of iron in deoxyhemoglobin
Iron slightly outside
porphyrin plane
His (imidazole ring)
binds 5th
coordination site
6th site for O2 binding
O2 binding, conformational change
Iron moves into plane, his is pulled along
Quaternary structure of hemoglobin
Pair of identical
alpha-beta dimers
Transition from T-to-R state in hemoglobin
Interface most affected
As O2 binds, top pair rotate 15o with respect to bottom pair
Oxygen affinity of fetal v maternal red blood cells
Fetal Hgl does not
bind 2,3-BPG,
higher O2 affinity
Fetal hemoglobin
tetramer has
2 alpha & 2 gama
chains,
Gene duplication
Isozymes of lactate dehydrogenase: glucose metabolism
Rat heart LDH isozyme profile changes with development
H(heart) isozyme (chain)= square, M(muscle) isozyme = circle
Tissue content of LDH
Functional LDH is tetrameric, with different combinations of subunits possible.
H4 (heart) has higher affinity for substrates than does M4 isozyme,
different allosteric inhibition by pyruvate
H4
H 3M
H2M2
HM3
M4
Some isozymes in blood indicative of tissue damage, used for clinical diagnosis
Increase in serum levels of H4 relative to H3M, indicative of
myocardial infraction (heart attack)
Examples of covalent modification
Phosphorylation widely used for regulation
Gamma
phosphoryl
group
Some known protein kinases
Protein phosphotases
Reverse the effects of kinases, catalyze hydrolytic removal of
phosphoryl groups attached to proteins
Activation by proteolytic cleavage
Secretion of zymogens by acinar cell of pancreas
Pancreas, one of the most
active organs in
synthesizing & secreting
proteins
Acinar cell stimulated by
hormonal signal or
nerve impulse, granule
content released into
duct to duodenum
Proteolytic activation of chymotrypsinogen
Active enzyme generated
by cleavage of a single
specific peptide bond
3 chains linked by 2
interchain disulfide
bonds, (A-B & B-C)
Conformations of chymotrypsinogen & chymotrypsin
Electrostatic interaction
between Asp 194
carboxylate & Ile 16
-amino group possible
only in chymotrypsin,
essential for activity
Zymogen activation by proteolytic cleavage
Zymogens orange, active enzymes yellow
Secreted by cells
that line duodenum
Digestive proteins of duodenum
Interaction of trypsin with its inhibitor
Lys 15 & Asp 189
form salt bridge
inside the active
site