ENZYMES KINETICS, INHIBITION, REGULATION

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ENZYMES KINETICS, INHIBITION, REGULATION
Muhammad Jawad Hassan Assistant
Professor Biochemistry
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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 xS/(S Km)
Michaelis-Menten Equation
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Steady-state pre-steady-state conditions
At pre-steady-state, P is low (close to
zero time), hence, V0 for initial reaction
velocity
At equilibrium, no net change of S P or of
ES E
At pre-steady state, we can ignore the back
reactions
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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
k2
k1
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
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Michaelis-Menten kinetics (summary)
Start with V0 k2ES, and derive, V0 Vmax
xS/(S Km) At low S (S lt Km), V0
(Vmax/Km)S At high S (S gt Km), V0
Vmax When S Km, V0 Vmax/2. Thus, Km
substrate concentration at which the reaction
rate (V0) is half max.
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Range of Km values
Km provides approximation of S in vivo for many
enzymes
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Lineweaver-Burk plot (double-reciprocal)
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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
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Enzyme inhibition
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A competitive inhibitor
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Methotrexate
A competitive inhibitor of dihydrofolate
reductase - role in purine pyrimidine
biosynthesis
Used to treat cancer
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Kinetics of competitive inhibitor
Increase S to overcome inhibition Vmax
attainable, Km is increased
Ki dissociation constant for inhibitor
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Competitive inhibitor
Vmax unaltered, Km increased
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Kinetics of non-competitive inhibitor
Increasing S cannot overcome inhibition Less E
available, Vmax is lower, Km remains the same for
available E
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Noncompetitive inhibitor
Km unaltered, Vmax decreased
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Enzyme inhibition by DIPF
Group - specific reagents react with R groups of
amino acids
diisopropylphosphofluoridate
DIPF (nerve gas) reacts with Ser in
acetylcholinesterase
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Affinity inhibitor covalent modification
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Catalytic strategies commonly employed

• 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.

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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
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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
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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
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Covalent catalysis
Hydrolysis in two stages
Deacylation to regenerate free enzyme
Acylation to form acyl-enzyme intermediate
Ser 195 OH group attacks the carbonyl group
Acyl-enzyme intermediate is hydrolysed
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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
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The catalytic triad (constellation of residues)
Ser 195 converted into a potent nucleophile, an
alkoxide ion
Imidazole N as base catalyst, accepts H
ion, positions polarizes Ser
Asp 102 orients His 57
H ion withdrawal from Ser 195 generates alkoxide
ion
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Regulatory Strategies Enzymes Hemoglobin

• 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

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Aspartate transcarbamoylase reaction
Committed step in pyrimidine synthesis inhibited
by end product CTP
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CTP inhibits ATCase
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CTP stabilizes the T state
CTP binds to regulatory subunits
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R and T states in equilibrium
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ATCase displays sigmoidal kinetics
Substrate binding to one active site converts
enzyme to R state increasing their activity
active sites show cooperativity
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Basis of sigmoidal curve
R T states equivalent to 2 enzymes with
different Kms
Cooperativity
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Effect of CTP on ATCase kinetics
CTP stabilizes the T state, curve shifts to right
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Effect of ATP on ATCase kinetics
ATP, allosteric activator, stabilizes R state,
curve shifts to left
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Oxygen delivery by hemoglobin, cooperativity
enhanced
98 - 32 66
63 - 25 38
Cooperativity enhances delivery 1.7 fold
Partial pressure of oxygen
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Heme group structure
4 linked pyrrole rings form a tetrapyrrole ring
with a central iron atom. side chains attached
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Position of iron in deoxyhemoglobin
Iron slightly outside porphyrin plane
His (imidazole ring) binds 5th coordination
site 6th site for O2 binding
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O2 binding, conformational change
Iron moves into plane, his is pulled along
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Quaternary structure of hemoglobin
Pair of identical alpha-beta dimers
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Transition from T-to-R state in hemoglobin
Interface most affected
As O2 binds, top pair rotate 15o with respect to
bottom pair
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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
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Isozymes of lactate dehydrogenase glucose
metabolism
Rat heart LDH isozyme profile changes with
development
H(heart) isozyme (chain) square, M(muscle)
isozyme circle
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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 H3M 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)
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Examples of covalent modification
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Phosphorylation widely used for regulation
Gamma phosphoryl group
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Some known protein kinases
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Protein phosphotases
Reverse the effects of kinases, catalyze
hydrolytic removal of phosphoryl groups attached
to proteins
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Activation by proteolytic cleavage
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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
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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)
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Conformations of chymotrypsinogen chymotrypsin
Electrostatic interaction between Asp
194 carboxylate Ile 16 ?-amino group
possible only in chymotrypsin, essential for
activity
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Zymogen activation by proteolytic cleavage
Zymogens orange, active enzymes yellow
Secreted by cells that line duodenum
Digestive proteins of duodenum
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Interaction of trypsin with its inhibitor
Lys 15 Asp 189 form salt bridge inside the
active site
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