Enzyme Kinetics

MICHAELIS-MENTEN KINETICS: LINEWEAVER-BURK PLOTS

 L-B equation converts M-M hyperbola into a straight line
o Inverse of a hyperbola is a straight line
o Straight lines are easier for humans to interpret than hyperbolas
 Generalization:
a∙x
o Equation for a hyperbola: y=
c ∙ x +b
1 c b
o Inverse of a hyperbola: = +
y a a∙ x
o plot of 1/x vs. 1/y is a straight line with a Y-intercept of a c/a and a slope of b/a
o apply to M-M equation
M-M EQUATION
Hyperbola straight line = a + bx
a∙x
y=
c ∙ x +b
V max ∙(S)
v 0=
(S )+ K M
L-B EQUATION
1/hyperbola = straight line
1 c b
= +
y a a∙ x
1 1 KM
= +
v 0 V max V max ∙(S )
 plot 1/V0 vs. 1/(s)
 y intercept = 1/Vmax
 slope = KM/Vmax
M-M PLOT
L-B PLOT

 KM=0.5 mM; Vmax = 1 μM/sec; (S) units are mM; v0 units are μM/sec.
 Y intercept: 1/Vmax 

ENZYME INHIBITION
 Reversible inhibitors bind to and dissociate from the enzyme
o Often structural analogs of substrates or products
o Often used as drugs to slow down a specific enzyme
 A reversible inhibitor can bind to the:
o Free enzyme and prevent binding of the substrates and/or
o Enzyme-substrate complex and prevent formation of product
 Irreversible inhibitors usually from a covalent bond with the enzyme and hence stay
bound to the enzyme
o One inhibitor molecule can permanently shut down an enzyme molecule
o Often either toxins or pharmaceutical drugs
COMPETITIVE INHIBITION

V max∙(S)
v 0=
α ∙ K M +(S )

 Has a structure similar to but different than the substrate that fools the enzyme
 Binds to the site instead of the substrate because both cant bind at the same time
 Increase substrate concentration to overcome competitive inhibitor
M-M PLOT FOR COMPETITIVE INHIBITION

L-B PLOT FOR COMPETITIVE INHIBITION

  1/initial velocity & 1/substrate concentration
 As the concentration of the inhibitor increases, the curves go up instead of down
o As V0 moves down (becomes less), 1/V0 moves up (becomes greater)
 All three lines converge at the Y-intercept (1/Vmax)
 As 1/(S) approaches 0, V0 approaches Vmax weather it’s in the presence of an inhibitor or
not
COMMENTS
 Competes w/ the substrate for biding to the enzyme
o Both bind at active site
o Does not affect catalysis, only binding of substrate to the enzyme
 No change in Vmax; apparent increase in KM
 Its L-B plot lines intersect at the y-axis

FIRST STEPS IN THE SYNTHESIS OF CHOLESTEROL

 HMG-CoA is a unique enzyme in steroid biosynthesis

 Used to lower cholesterol
o As a competitive inhibitor of HMG-CoA
STRUCTURAL FORMULAE OF TWO STATIN INHIBITORS & OF THE ENZYME
SUBSTRATE HMG-CoA

 HMG Co-A is the structure of the substrate for the enzyme HMG Co-A reductase
 Compactin and simvastatin are competitive inhibitors of HMG-CoA reductase
o Help lower cholesterol

M-M MODEL OF 3 TYPES OF REVERSIBLE INHIBITION

 an enzyme inhibitor can be characterized by its 2 dissociation constants, Ki and Ki’

o Competitive: K'i = ∞ (i.e., no ESI forms) i.e., 1 + (I)/Ki' = 1
o Uncompetitive: Ki = ∞ (i.e., no EI forms) i.e., 1 + (I)/Ki = 1
 Inhibitor can only bind the enzyme substrate complex
o Mixed: Ki ≠ K'i i.e., 1 + (I)/Ki does not equal 1 + (I)/Ki'
 Non-competitive: Ki = K'i (special case of mixed inhibition, rare in Nature)
  model for all kinds of mixed inhibition
& competitive inhibition

MIXED INHIBITION

 Two binding sites

o One for the substrate and a separate site for the inhibitor
 Inhibitor can bind either to the free enzyme OR to the enzyme substrate complex
L-B PLOT FOR MIXED INHIBITION
  As the inhibition concentration increases, KM increases
 The inhibitor decreases the Vmax
 Inhibitor increases KM
o takes more substrate to get to a certain velocity in the presence of an inhibitor 
COMMENTS
 Binds either free enzyme or enzyme-substrate complex
o Binds to the regulatory site
 Inhibits both substrate binding and catalysis
 Results in Vmax and apparent increase in KM
 Its L-B lines intersect left of the y-axis
o Above the x-axis if Ki < Ki'
o Below the x-axis if Ki > Ki'
o On the x-axis if Ki = Ki' (= non-competitive inhibition, which has never been
observed)
UNCOMPETITIVE INHIBITION
  Inhibitor can bind only to the enzyme substrate complex, not the free enzyme
o When a ligand binds to a protein (such as a substrate to an enzyme), there is a
change in conformation
o In some cases, a site that is “closed” can open up and bind an inhibitor
 Inhibitor can only bind when its site opens up after the substrate binds to the enzyme
L-B PLOT FOR UNCOMPETITIVE INHIBITION

COMMENTS
 Only binds to ES complex
o Does not affect substrate binding
o Inhibits catalytic function
 Results in a decrease of Vmax & apparent decrease of KM
 Does not change KM/Vmax
 Its L-B lines are parallel
GLYPHOSATE INHIBITION OF 5-ENOLPYRUVYLSHIKIMATE-3PHOSPHATE
SYNTHASE (EPSPS)
 Glyphosate = roundup (weed killer)
o An uncompetitive inhibitor of ESPS because it can inhibit only if shikimate-3-P is
first bound to the enzyme
 ESPS
 o enzyme not found in animals
o Required for plant growth
o Have to make Phe, Tyr, & Trp to make proteins

FINAL COMMENTS ON M-M KINETICS

 Mixed and uncompetitive inhibitions occur essentially only with enzymes that have 2
(rarely 3) substrates
o Mixed inhibition indicates one type of 2-substrate catalytic mechanism (ternary
complex intermediate), uncompetitive inhibition another mechanism (ping-pong)
 Many (most?) real-life inhibitions are irreversible
o E.g. organofluorophosphates inhibit enzymes with the catalytic triad (one
example: sarin and acetylcholinesterase)

(ACETYL)CHOLINESTERASE

 Organofluorophosphates inhibit cholinesterase which inactivates the neurotransmitter

acetylcholine
 This esterase has the same catalytic mechanism as serine proteases
 Nerve poison (sarin) used by Sadam Hussein, Amun Shinryko and Bashar al-Assad

ANOTHER VIEW OF ENZYMES W/ THE CATALYTIC TRIAD (S-H-D) BEING

INHIBITED BY ORGANOFLUOROPHOSPHATES
HEAVY METAL POISONING
Enz-SH + M+  Enz-S-M + H+
 E.g.: Pb, Hg, Cd, Ag, etc
o Can bind any available sulfhydryl group (on Cys residues)
o May or may not be near the active site
o Erythrocytes and CNS especially vulnerable
o Essential irreversible inhibition
 E.g. Silver Valley, ID
 E.g. major locus of lead poisoning: porphobilinogen synthase
o Early step in biosynthesis of heme
o Leads to anemia
PORPHOBILINOGEN SYNTHASE (= ALA DEHYDRATASE)

 5-aminolevulinate is the first common (= unique) precursor of heme-like compounds

BIOSYNTHESIS OF HEME
  5-aminolevulinate in the red box
o Early step in synthesis of heme
o If inhibited, then the whole synthesis is stopped and the organism (cells) are in
trouble because RBC and the CNS are especially sensitive to this inhibition
 Leads to anemia because RBC count is low

ASSUMPTION VS. REALITY

 Although theoretically examples of true uncompetitive and noncompetitive inhibitors, in
reality, observed kinetics are not that simple
o Uncompetitive inhibition: inhibitor binding should occur only if the active site is
occupied by substrate. But most (all?) cases, the inhibitor will have some affinity
for the unoccupied enzyme as well
o Noncompetitive inhibition: the inhibitor affinity should be unchanged regardless
of whether substrate is bound or not. In fact, the affinity for the inhibitor
(always?) changes when substrate is bound
 True competitive inhibition is common
 In reality, we see competitive and mixed inhibition only

INHIBITION SPECTRUM: NO ESI  NO EI

 Inhibition found in nature shown in red

ENZYME REGULATION CAN BE VIA:

 Noncovalent modification
o Often readily reversible
 Covalent modification
o Often reversible unless another enzyme becomes involved