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Enzyme Kinetics

I.
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Reaction Kinetics
Rate equations to describe
progress of first-order and
second-order reactions
Michaelis-Menten eqn: Vi ↔ Vmax,
Km
Overall catalytic efficiency =
kcat/Km
Lineweaver-Burk plot: present
data and calculate Km, Vmax
Bisubstrate Reactions can be
Ordered/Random/Pingpong
mechanism

Chemical Kinetics described by
Rate Equations
Reaction Order = number of
molecules
participating in Elementary reaction
- reaction velocity ≈
concentration of
reactant (first-order reaction)
- number of molecules that
must collide
to form a product
- 2 molecules needed to form a
product(second-order reaction)
Enzyme Kinetics follow MichaelisMenten Eqn
At ↑↑S, reaction rate becomes

independent of substrate
concentration (zeroth order)
- k2 becomes rate-limiting step
M-M Equation assumes ES as steady
state
- k-1 >> k1
- Since [S]>>[E], [ES] assumed to
be
constant until [S] depleted
(steady state)

- Michaelis constant, Km = (k-1 +
k1)/k1
- vo = Vmax[S]/(Km + [S])
- Vmax occurs at high [S] or [E] is
saturated
- Km is [S] where reaction rate =
½Vmax
- small Km = maximal catalytic
efficiency at
low [S]
kcat/Km as measure of catalytic
efficiency
- kcat = Vmax/[E] – number of reaction
processes that each active site
catalyzes/unit
time
- a second order reaction
- when [S] << Km, little [ES] is
formed
- rate of reaction varies directly on
how often
S and E encounter each other
- upper limit: k1
- Rate of decomposition of ES → E
+ P not
greater than rate of E + S → ES

1st substrate bind with enzyme II.possibility of other intermediates Bisubstrate Reactions Involve two substrates to form two products .substrates combine with enzyme before reaction may occur = sequential reaction 1.no order of preference for substrate addition Nevertheless. or transfer of specific functional groups . ↑/↓Km .one of more products released before all substrates have been added . steady state fails to establish the true mechanism .substrates A and B don’t encounter at the enzyme surface Sequential Reactions via Single Displacement . Ordered Mechanism . vo ≈ Vmax asymptotically 2.Kinetic Data provide Vmax and Km At ↑S.oxidation-reduction. - - → to form binding site for 2nd substrate - Enzyme Inhibition Inhibitors interact reversibly/irreversibly to change Km and/or Vmax Competitive IN: binds with active site.compulsory substrate addition to enzyme . Random Mechanism .both binding sites present in free enzyme Ping Pong reactions . ↑Km Uncompetitive IN: ↓Km ↓Vmax Mixed enzyme inhibitor: ↓Vmax.

products formed may bind at the active site.directly competes with normal substrate at the active site e.control in cell activity Transition state analogs .compound mimics TS .Inhibitors .tightly binds with enzyme and permanently block activity Competitive Inhibition . accumulate and compete with substrate .g.substances that reduce enzyme’s activity: a) Irreversible .effective catalysis dependent on enzyme’s ability to bind and stabilize TS . malonate inhibits succinate dehydrogenase Product inhibition .

metal ions may act as INNC .Allosteric effectors bind with multisubunit enzymes → cooperative conformation change → ∆catalytic activity .substrate binding doesn’t affect binding of inhibitor . Km unchanged than it is .require that inhibitor affect the Irreversible Inhibitor .Phosphorylation ↔ Dephosphorylation .addition of substrate won’t affect the effect . versus methanol → slowing production of formaldehyde → methanol excreted through urine Uncompetitive Inhibition Inhibitor binds directly to ES complex but not to free enzyme .α = 1 + [I]/KI .INC decreases the free [E] for substrate binding ..if INNC binds to E and ES equally: only Vmax affected .resemble pure noncompetitive inhibitor = decreasing free [E] at all [S] III.however.presence of [I] make [S] smaller catalytic nature of the enzyme and not formation of ES complex Mixed (noncompetitive) inhibition Inhibitor binds to both free E and ES complex . Control of Free Enzyme . Ethanol competes at active site.distorts the active site → inactivated enzyme . vo ≈ Vmax *Principle behind use of ethanol for methanol poisoning.vo = Vmax[S]/(αKm + [S]) . at ↑↑[S].

covalent modification.induce control by shifting to more/less active conformation cooperatively Enzyme must be able to control and coordinate its activity depending on its metabolic need Ways to control: 1.modulation of activity by structural alteration that influence ES complex formation or turnover number . CTP dissociates from ATCase ↑CTP synthesis .both substrate bind . a feedback inhibitor.coordination of ATP and CTP in purine and pyrimidine synthesis .allosteric effectors that cause changes in enzymatic activity . ↓CTP synthesis . usually phodephosphorylation of specific Ser. inhibits its earlier step in its biosynthesis . Enzyme Activity .controlled by cell and dramatic changes over time 2. Enzyme Availability .allosterically inhibited by cytidine triphosphate (CTP). purine nucleotide .allosterically activated by adenosine triphosphate (ATP).[ATP]>[CTP] → ATCase activated to form pyrimidine nucleotides until equal . aspartate carbamoylase (ATCase) . Tyr residues Allosteric Control by binding at site not the active site e. Thr.amount of enzyme depend on its synthesis ↔ degradation .[CTP]>[ATP] → permits ATP synthesis .↓[CTP].CTP.g.↑[CTP]. pyrimidine nucleotide .catalyzes N-carbamoyl aspartate → carbamoyl phosphate + aspartate .

CTP: T-state binding and ↑ stability . Allosteric effectors lead to structural changes .ATP: R-state binding and ↑ stability . ↑ ATP.rate controlling step in glycogen breakdown for fuel in metabolic processes Phosphorylation-dephosphorylation resemble allosteric control Glycogen phosphorylase . glucose concentration (thus no need for glycogen breakdown) ↑muscle excertion.Both CTP and ATP bind to same site . glycogen phosphorylase .glucose as only allosteric effector . upon conversion of G1P) prefers binding with T state of pb → inactivating enzyme . less active) → reorientation of active site → decrease in activity . G6P.T state: inactive = malformed active site and surface loop (residues 282284) blocking active site from substrate .AMP prefers binding with R state of pa → activating enzyme ↓muscle excertion.binds to T-state → inactivating enzyme phosphorylase b (pb) = dephosphorylated form .catalyzes phosphorolysis (bond cleavage + phosphate group substitution) of glycogen to yield glucose-1phosphate (G1P) . .ATP + T-state → trimer move apart (like Rstate) →→ increase in activity Control by Covalent Modification (Phosphorylation) .to balance out pyrimidine-purine conc. ↑AMP (induce breakdown to help fuel metabolism) In phosphorylase a.processes catalyzed by protein kinases and protein phosphatases e.g.ATP. glucose-6-phosphate (G6P.CTP + R-state → contraction of regulatory dimer (similar to T-state.R state: active = Arg 569 oriented to allow binding with substrate and 282-284 no longer blocks active site Phosphorylation of Ser 14 promotes T(inactive)→R(active) conformational change phosphorylase a (pa) = phosphorylated form + phosphoryl group esterified to Ser 14 .

protein kinases/phosphatases linked in series .greater capacity at signal amplification because small fractional change in effector concentration cause larger fractional enzymatic activity .Ser 14 phosphate group forms ion pairs with two cationic Arg side chains → linking active site. subunit interface.. N- terminal region → glycogen phosphorylase’s helices pull apart → triggers quaternary T to R transition * Ser 14 acts as internal allosteric effector to shift T ↔ R equilibrium toward R state Cascades of Protein Kinases = Greater sensitivity .