Enzymes -Properties

With the exception of a few catalytic RNA molecules

(ribozymes ) majority of enzymes are proteins

The catalytic activity depends on the integrity of their native

protein conformation

1
International classification of Enzymes
No Class Type of reaction catalyzed
1 Oxidoreductases Enzymes catalyzing oxidoreductions between
two substrates, S and S’
S reduced + S’ oxidized = S oxidized + S’ reduced
2 Transferases Enzymes catalyzing a transfer of a group, G (other than
hydrogen), between a pair of substrates S and S’
S-G + S’ = S’–G + S
3 Hydrolases Enzymes catalyzing hydrolysis of ester, ether, peptide,
glycosyl, acid anhydride, C-C bonds
4 Lyases Enzymes catalyzing addition of groups to double bonds, or
formation of double bonds by removal of groups
X Y

C ─ C = X-Y + C C
5 Isomerases Enzymes catalyzing transfer of groups within molecules to
yield isomeric forms
6 Ligases Enzymes catalyzing formation of C-C, C-S, C-O, and C-N
bonds by condensation reactions coupled to ATP hydrolysis

2
Effective and highly specific catalysts
Highly efficient
• Enhance the rates of the corresponding noncatalyzed reactions by at least
106
• Neither consumed nor permanently altered as a consequence of the
reaction
Selective
• Specific for the type of reaction they catalyze
• The ability of an enzyme to catalyze one specific reaction and essentially no
others is perhaps its most significant property
• Specific for a single substrate or a small set of closely related substrates
Optical specificity-
• show absolute optical specificity for at least a portion of the substrate
molecule
– glycolytic pathway enzymes catalyze interconversions of D- but not L-
phosphosugars
– most mammalian enzymes act on L- but not D- amino acids
Group specificity-
• Lytic enzymes act on specific chemical groupings eg pepsin and trypsin on
peptide bonds
– a large number of substrates may be attacked, thus lessening the number of
digestive enzymes that might be required
3
• Certain lytic enzymes exhibit a higher order of group specificity
– Chymotrypsin preferentially hydrolyzes peptide bonds in which the carboxyl
group is contributed by the aromatic amino acids phenyalanine, tyrosine and
tryptophan
– Carboxypeptidase and aminopeptidase split off amino acids one at a time from
the carboxyl- and amino-terminal end of polypeptide chains respectively
– Although some oxidoreductases utilize either NAD + or NADP+ as electron
acceptor most use exclusively one or the other
• Specificity enables cells to simultaneously conduct and independently control a
broad spectrum of biochemical processes

Prosthetic groups, cofactors, and coenzymes play important roles in

catalysis
• Many enzymes contain small nonprotein molecules and metal ions that participate
directly in substrate binding or catalysis
• Prosthetic groups, cofactors, and coenzymes increase catalytic capability beyond
those afforded by the limited number of functional groups present on the
aminoacyl side chains of polypeptides

4
(i) Prosthetic groups are coenzymes or metal ions that are tightly integrated into an
enzyme’s structure
• Tightly incorporated into a protein’s structure by covalent and noncovalent forces
– E.g. pyridoxal phosphate, flavin mononucleotide (FMN), flavin adenine
dinucleotide (FAD), thiamin pyrophosphate, biotin, and metal ions of cobalt,
copper, magnesium, manganese, selenium, and zinc
– Metal ions are the most common prosthetic groups
• About a third of the enzymes with tightly bound metal ions are metalloenzymes (metal
ion retained during purification process)
• Metal ions
– in redox reactions generally are complexed to prosthetic groups such as heme
or ion sulphur clusters
– may facilitate binding or orientation of substrates, formation of bonds with
reaction intermediates, or interaction with substrates to render them more
electrophilic (electron-poor) or nucleophilic (electron-rich)
– nearly a third of all known enzymes require one or more metal ions for
catalysis
(ii) Cofactors (Activators)
• Serve similar functions as prosthetic groups
• bind transiently and reversibly to the enzyme or substrate
• must be present in the medium surrounding the enzyme for catalysis to occur
• most common cofactors are metal ions
• enzymes that require metal cofactors are metal-activated enzymes
5
(iii) Coenzymes ( complex organic or metalloorganic molecules) serve as substrate
shuttles
• Recyclable shuttles or group transfer reagents
– transport substrates from their point of generation to their point of
utilization eg
• methyl groups (folates),
• acyl groups (coenzyme A),
• oligosaccharides (dolichol)
– stabilize substrates

• Enzymes that require coenzymes are in classes 1, 2, 5 and 6

• Lytic reaction, including hydrolytic reactions such as those catalyzed by digestive
enzymes, do not require coenzymes (Classes 3 and 4)

• A complete, catalytically active enzyme together with its bound coenzyme and/or
metal ions is called a holoenzyme
• The protein part of an enzyme is called the apoenzyme or opoprotein

6
Examples of some inorganic ions that serve as cofactors for enzymes

Ions Enzymes
Cu 2+ Cytochrome oxidase

Fe 2+ or Fe 3+ Cytochrome oxidase, catalase

peroxidase
K+ Pyruvate kinase

Mg 2+ Hexokinase, glucose 6-phosphatase

Pyruvate kinase

Se Glutathione peroxidase

Zn 2+ Carbonic anhydrase
Alcohol dehydrogenase

7
8
(iv) Many coenzymes, cofactors and prosthetic groups are derivatives of B vitamins
-Water-soluble B vitamins supply important components of numerous coenzymes
Coenzyme Examples of chemical Dietary precursor in mammals
groups transferred
Biocytin CO 2 Biotin
Coenzyme A Acyl groups Pantothenic acid
5’-deoxyadenosyl H atoms and alky Vitamin B 12
cobalamin groups
Flavin adenine Electrons Riboflavin (Vitamin B 2)
dinucleotide (FAD)
Lipoate Electrons and Not required in diet
acyl groups
Nicotinamide adenine Hydride ion (:H ─) Nicotinic acid (Niacin)
dinucleotide (NAD)
Pyridoxal phosphate Amino acids Pyridoxine (Vitamin B 6)
Tetrahydrofolate One-carbon groups Folate
Thiamine pyrophosphate Aldehydes Thiamine (Vitamin B 1)

-many in addition contain the adenine, ribose, and phosphoryl moieties of AMP or ADP
e.g. NAD, NADP, FAD,

9
Catalysis occurs at the active site
-Cleft or pocket
- The catalytic sites of multimeric enzymes are often located at the interface between
subunits and recruit residues from more than one monomer
-The three-dimensional active site – shields substrate from solvent
- facilitates catalysis
- Substrate binds to the active site
-aligns portions of the substrate that will undergo change with the
chemical functional groups of aminoacyl residues
- Active site also binds and orients cofactors or prosthetic groups
- Many amino acyl residues drawn from portions of the polypeptide chain contribute to
the extensive size and three-dimensional character of the active site
Some common features of active sites of enzymes
1.Active site takes up a relatively small part of the total volume of the enzyme
2.Three dimensional entity formed by groups from different parts of the linear amino
acid sequence
3.Substrates are bound to active site by multiple weak interactions
4.Active sites are clefts or pockets. Water is usually excluded unless it’s a reactant
5.The specificity of the binding depends on the precisely defined arrangement of the
atoms in an active site

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Substrates induce conformational change
-‘Lock and key model’ by Emil Fisher accounted for the specificity of enzyme-
substrate interactions but failed to account for the dynamic changes that
accompany catalysis
. The active site of the unbound enzyme is complimentary to that
of the substrate
-The induced fit model by Daniel Koshland (confirmed by biophysical studies
during substrate binding)
.When a substrate approaches and binds to an enzymes it induces a
conformational change in the enzyme that is complimentary to that of
the substrate
.The enzyme then induces reciprocal changes in the substrate to facilitate
transformation of substrate into products

Isoenzymes are distinct enzyme forms that catalyze the same reaction
-Physically distinct versions of a given enzyme catalyzing the same reaction
-Isoenzymes arise through gene duplication
-May exhibit subtle differences in sensitivity, regulatory cofactors, substrate
affinity that adapt them to specific tissues or circumstances
-Some may enhance survival by providing a backup copy of the essential
enzyme

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The analysis of certain enzymes aids in diagnosis
• Enzymes that fulfil functions indispensable to cell viability are present
through out body tissues
• Other enzymes or isoenzymes are expressed only in specific tissues, during
certain periods of development, or in response to specific physiological and
pathophysiological changes
• Analysis of the presence and distribution of enzymes and isoenzymes-
whose expression is normally tissue-, time- or circumstances-specific often
aids diagnosis
(i) Nonfunctional plasma enzymes aid diagnosis and prognosis
• Certain enzymes, proenzymes, and their substrates are present at all times
in the circulation of normal individuals and perform physiologic functions in
blood
– Known as functional plasma enzymes e.g.
• lipoprotein lipase, pseudo cholinesterase, proenzymes of blood coagulation
and blood clot dissolution
• Majority are synthesized and secreted by the liver
• Plasma also contain other enzymes of no known physiological function in
blood

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• Known as nonfunctional plasma enzymes
– Arise from normal destruction of erythrocytes, leukocytes, and other tissues
– tissue damage or disease is generally accompanied by increase in the levels of
several nonfunctional plasma enzymes

(ii) Isoenzymes of lactate dehydrogenase are used to detect myocardial

infarctions
L-lactate dehydrogenase is a tetrameric enzyme whose four subunits occur in
two isoforms i.e. H (for heart) and M (for muscle)

Lactate dehydrogenase Subunit

isoenzyme
I1 HHHH
I2 HHHM
I3 HHMM
I4 HMMM
I5 MMMM

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• The heart expresses the H subunit exclusively; isoenzyme I1 predominates.
• The I5 isoenzyme predominates in the liver
• Small quantities of lactate dehydrogenase are normally present in the
plasma
• Following myocardial infarction or liver disease the damaged tissue releases
the characteristic lactate dehydrogenase into the blood

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Serum Enzyme Major diagnostic use

Amino transferases
Aspartate amino transferase (SGOT) Myocardial infarction
Alanine amino transferase (SGPT) Viral hepatitis

Amylase Acute pancreatitis

Ceruloplasmin Hepatolenticular degeneration

(Wilson’s disease)
Creatine kinase Muscle disorders and myocardial
infarction
γ-Glutamyl transpeptidase Various viral diseases

Lactate dehydrogenase Myocardial infarction

(isoenzymes)
Lipase Acute pancreatitis
Phosphatase, acid Metastatic carcinoma of the
prostate
Phosphatase, alkaline Various bone disorders, obstructive
(isoenzyme) liver disease

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Enzyme Kinetics
•Kinetics is the branch of science concerned with the rates of chemical reactions.
•The study of enzyme kinetics addresses the biological roles of enzymatic catalysts and how they
accomplish their remarkable feats.
•In enzyme kinetics, the maximum reaction velocity that the enzyme can attain is determined and its
binding affinities for substrates and inhibitors.

•Assuming that is an elementary reaction and that it is spontaneous and essentially irreversible
– The velocity, v, or rate, of the reaction A→P is the amount of P formed or the amount of A
consumed per unit time, t. That is,

– For this case, the rate law is

– The rate is proportional to the concentration of A, and k is the proportionality constant, or rate constant

16
• V is a function of [A] to the first power, or, in the terminology of kinetics, v is first-order
with respect to A.
• The number of molecules that must simultaneously interact is defined as the
molecularity of the reaction- in the above case its 1 (unimolecular reaction)

• Consider the more complex reaction, where two molecules must react to yield
products

• Assuming this reaction is an elementary reaction, its molecularity is 2; that is, it is a

bimolecular reaction
• The velocity of this reaction can be determined from the rate of disappearance of
either A or B, or the rate of appearance of P or Q:

• The rate law is

• The rate is proportional to the concentrations of both A and B. Because it is
proportional to the product of two concentration terms, the reaction is second-order
overall, first-order with respect to A and first-order with respect to B.

17
• Molecularities greater than two are rarely found (and greater than
three,never)

18
Free Energy of Activation and the Action of Catalysts
•In a first-order chemical reaction, the conversion of A to P occurs because, at any
given instant, a fraction of the A molecules has the energy necessary to achieve a
reactive condition known as the transition state.
•This transition state sits at the apex of the energy profile in the energy diagram
describing the energetic relationship between A and P
•The average free energy of A molecules defines the initial state and the average free
energy of P molecules is the final state along the reaction coordinate.
•The rate of any chemical reaction is proportional to the concentration of reactant
molecules (A in this case) having this transition-state energy
•The higher this energy(transitional) is above the average energy, the smaller the
fraction of molecules that will have this energy, and the slower the reaction will
proceed
•The height of this energy barrier is called the free energy of activation, G‡
•Therefore free energy of activation, G‡, is the energy required to raise the average
energy of one mole of reactant (at a given temperature) to the transition-state energy.

19
• The relationship between activation energy and the rate constant of the reaction, k, is given by
the Arrhenius equation,

• where A is a constant for a particular reaction (not to be confused with the reactant species, A,
that we’re discussing).

• Another way of writing this is

• That is, k is inversely proportional to . Therefore, if the energy of activation

decreases, the reaction rate increases.

Decreasing G‡ Increases Reaction Rate

• There are two general ways that rates of chemical reactions may be accelerated
– First, the temperature can be raised. This will increase the average energy of reactant molecules,
which in effect lowers the energy needed to reach the transition state
– Second, the rates of chemical reactions can also be accelerated by catalysts. Catalysts work by
lowering the energy of activation rather than by raising the average energy of the reactants

20
• Two aspects of catalysts are worth noting:
– they are regenerated after each reaction cycle (AnP), and so can be
used over and over again; and
– catalysts have no effect on the overall free energy change in the
reaction, the free energy difference between A and P

21
Energy diagram for a chemical reaction (A→P) and the effects of (a)
raising the temperature from T1 to T2 or (b) adding a catalyst. Raising the temperature
raises the average energy of A molecules, which increases the population of A molecules
having energies equal to the activation energy for the reaction, thereby increasing the
reaction rate. In contrast, the average free energy of A molecules remains the same in
uncatalyzed versus catalyzed reactions (conducted at the same temperature). The effect of
the catalyst is to lower the free energy of activation for the reaction.

22
Kinetics of Enzyme-Catalyzed Reactions
•In analysis of enzyme-catalyzed reactions involving only a single substrate. At
low concentrations of the substrate S, v is proportional to [S], as expected for a
first-order reaction.
•However, v does not increase proportionally as [S] increases, but instead
begins to level off.
•At high [S], v becomes virtually independent of [S] and approaches a maximal
limit. The value of v at this limit is written Vmax.
•Because rate is no longer dependent on [S] at these high concentrations, the
enzyme-catalyzed reaction is now obeying zero-order kinetics

23
Substrate saturation curve for an enzyme-catalyzed reaction. The amount of
enzyme is constant, and the velocity of the reaction is determined at various
substrate concentrations. The reaction rate, v, as a function of [S] is described by
a rectangular hyperbola. At very high [S], v is Vmax. That is, the velocity is
limited only by conditions (temperature, pH, ionic strength) and by the amount of
enzyme present; v becomes independent of [S]. Such a condition is termed zero-
order kinetics. Under zero-order conditions, velocity is directly dependent on
[enzyme]. The substrate is bound at the active site of the enzyme.
24
• The physical interpretation is that every enzyme molecule in the reaction
mixture has its substrate-binding site occupied by S

25
 Plus
enzyme

Product
No
enzyme seconds hours

• The amount of product formed is the same in the absence or presence of enzyme but
the amount of product formed in seconds when the enzyme is present might take
hours or years to form in the absence of enzyme
– Product concentration levels off with time when reaction reaches equilibrium
– While S is being converted to P, P is also being converted to S at the same rate
such that the amount of P remains constant
– Therefore enzymes accelerate attainment of equilibrium but do not change the
position of equilibrium

26
• Activation energy barriers are crucial to life itself
-stability of macromolecules increases with the height of its activation
barriers
-without such barriers, macromolecules would revert spontaneously
to simple molecular forms
-enzymes have evolved to lower activation barriers selectively

27
Important features at equilibrium:
• Equilibrium constant is the ratio of the reaction constants
(not the reaction rates)
• The reaction rates ( not the rate constants) of the forward and the reverse
reactions are equal.
• It’s a dynamic state; individual substrates and products are continually being
interconverted
• The numerical value of the equilibrium constant Keq can be calculated
either from the concentrations of substrates and products at equilibrium or
from the ratio of k1/k-1

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Multiple factors affect the rate of enzyme catalyzed
reactions
These include, temperature, hydrogen ion concentration, substrate concentration, end
products, allosteric factors, covalent modification, hormones etc
Temperature
• Rising temperature increases the rate of both uncatalyzed and enzyme-catalyzed
reactions
-increase kinetic energy of the reactants
-increase the collision frequency of the reactants
• Heat energy can also increase kinetic energy of the enzyme to the point of disrupting
the noncovalent interactions that maintain the enzyme’s three dimensional structure
-polypeptide chain unfolds or denatures
-loss of catalytic activity
• The temperature range over which an enzyme maintains a stable , catalytically
competent conformation depends upon (and typically moderately exceeds) the normal
temperature of the cells in which it resides
-in humans enzymes generally exhibit stability at temperatures up
to 45 – 55oC
-in thermophilic microorganisms that reside in volcanic hot springs
enzymes may be stable at 100oC

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• Q10, or temperature coefficient , is the factor by which the rate of a biologic process
increases for a 10oC increase in temperature.
• Over the range in which the enzymes are stable, the rates of most biologic systems
double for a 10oC increase in temperature (Q10=2)
Hydrogen ion concentration
• Almost all enzyme-catalyzed reactions exhibit a significant dependence on hydrogen
ion concentration
• Most intracellular enzymes exhibit optimal activity at pH values between 5 and 9
• At high or low pH the enzyme is denatured as the changes in pH affects the charged
state of the enzyme, the substrates or both.
-enzymes whose mechanism depends on acid-base catalysis, the
residues involved must be in the appropriate state of protonation for
the reaction to proceed
-the binding and recognition of substrate molecules with dissociable
groups also typically involves the formation of salt bridges with
the enzymes
• The most common charged groups are the negative carboxylate groups and the
positively charged groups of protonated amines
• Gain or loss of charged groups thus will affect substrate binding and hence retard
catalysis

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Enzyme concentration
• Measurements of the rates of enzyme-catalyzed reactions employ short
periods which approximate initial rate conditions
-traces of products accumulate
-rate of the reverse reaction is negligible
• The initial velocity (vi) of the reaction is essentially that of the rate of the
forward reaction
• If a large molar excess of substrate over enzyme is employed
- vi is directly proportional to the concentration of the enzyme
-measurement of the initial velocity permits one to estimate the
concentration of enzyme in the biological sample
- but as stated above enzyme concentration has no effect on
Keq and ΔGo

31
Substrate concentration affects reaction rate
• The approach to understanding enzyme mechanism is to determine the rate of the
reaction and show changes in response to changes in experimental parameters
• Follow the increase in reaction product as a function of time
• Because [S] changes during reaction as substrate is converted to product, the initial
rate (Initial velocity) is the is the one that is measured
• In a typical reaction [S] is much higher than [E]. Therefore if only the beginning of the
reaction is considered, [S] changes can be considered insignificant and therefore
regarded as constant
• Vo can then be explored as a function of [S]
• Rate of the reaction, Vo is measured when there is negligible product formation and
thus no reverse reaction ie k-2 [P] ≈ 0 See below

k1 k2 k1 k2
E+S ES E + P becomes E+S ES E+P
k-1 k-2 k-1

32
Vmax .C
Vmax/2

vi .B
.A Vmax/2

[S]
Km

Effects of substrate concentration on the initial velocity of an

enzyme-catalyzed reaction

33
-As substrate concentration is increased , the vi increases until it reaches a
maximum value Vmax.
-When further increases in substrate concentration do not further increase vi
the enzyme is ‘saturated’ with substrate
-Curve is hyperbolic
-At points A or B increasing or decreasing substrate concentration will increase
or decrease the ES complexes with a corresponding change in vi
-At point C, essentially all the enzyme is present as the ES complex
-no free enzyme remains
-further increase in [S] can not increase the rate of the reaction
-Case B is where half the enzyme molecules are ‘saturated with’ substrate and
the velocity is accordingly half the maximal velocity (Vmax/2) at a particular
enzyme concentration

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The Michaelis-Menten equation models the effects of substrate
concentration

vi= Vmax[S]
Km + [S]
-illustrates in mathematical terms the relationship between initial reaction
velocity vi and substrate concentration [S]
The Michaelis constant Km is the substrate concentration at which vi is half the
maximal velocity (Vmax/2) attainable at a particular concentration of
enzyme
The relationship of initial reaction velocity on [S] and Km may be illustrated by
evaluating the equation under three conditions
(1) When [S] is very much less than Km (point A)
-the term Km + [S] is essentially equal to Km
-replacing Km + [S] with Km reduced the equation to
vi= Vmax[S] vi~ Vmax[S] ~ Vmax [S]
Km + [S] Km Km
-since Vmax and Km are constants,the ratio is a constant
-the initial reaction velocity is therefore directly proportional to [S]

35
(2) When [S] is so much greater than Km (point C)
-the term Km + [S] is essentially equal to [S]
- Replacing Km + [S] with [S] reduces the equation to
vi= Vmax[S] vi~ Vmax[S] vi~ Vmax
Km + [S] [S]
-when [S] greatly exceeds Km , the reaction velocity is maximal (Vmax) and
unaffected by the increases in substrate concentration

(3) When [S] = Km (point B)

vi= Vmax[S] = Vmax[S] = Vmax

Km + [S] 2[S] 2
- when [S] equals Km the initial velocity is half-maximal
- Km may be determined experimentally from the substrate concentration
at which the initial velocity is half-maximal

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• The [ES] is key to understanding the kinetic behaviour
– The enzyme binds substrate in a relatively fast manner
– The ES complex then breaks down in a slower second step to form product.
Because it is slow it must be rate limiting

E+S ES

• At low [S] most of the enzyme is in the uncombined E form. The rate is proportional to
[S] because the equilibrium of the above is shifted towards formation of ES as [S]
increase
• Vmax is observed when virtually all enzyme is present in the ES complex and [E] is small
• Vmax reveals the turn-over number of the enzyme ie the number of substrate molecules
converted to product by an enzyme molecule per unit time when the enzyme is fully
saturated
• The turn-over number is equal to the rate constant k2, (kcat)
• Vmax reveals the turn-over number of an enzyme if the concentration of the active sites
[Et] is known
Vmax = k2[Et] k2 = Vmax/ [Et]

37
A linear form of the Michaelis-Menten equation is used to
determine Km and Vmax

Rearranging the Michaelis-Menten equation gives

1 = Km 1 1
+
V Vmax [S] Vm

-This is the equation for a straight line, y = ax + b, where y = 1/vi and x= 1/[S]
-y intercept b, is 1/Vmax and slope a, is Km/Vmax. x intercept is -1/Km
-called a double reciprocal or Lineweaver-Burk plot

Slope = Km
1 Vmax
vi
1 Km is most easily calculated
─ 1
Km from the x intercept
Vmax

1
[S] 38
-The Lineweaver-Burk plot
-has the advantage of allowing a more accurate determination of Vmax,
which can only be approximated from a simple plot of vi Vs [S]
-also useful in distinguishing between certain types of enzymatic
reaction mechanisms and in analyzing enzyme inhibitions

The relationship of Km to Kd, the dissociation constant of the enzyme

substrate complex
• The affinity of the enzyme for its substrate is equal to the inverse of the
dissociation constant, Kd for ES
k1
E+S ES
K-1

k 1 i.e the less the tendency of the substrate and enzyme to

Kd= k1 dissociate , the greater is the affinity of the enzyme
The Km value of an enzyme for its substrate may also serve as a measure of its K d
if its assumed that the first step of the enzyme-catalyzed reaction for the formation
of the ES complex is fast and always at equilibrium; the rate of dissociation of ES
to E + S must be much faster than its dissociation to enzyme and product
39
 Vmax
• In the Michaelis-Menten expression, the [S] that gives vi  is
2
k 2  k 1
[S ]   Km
k1
k 1
But when k-1 >> k2 , then k2 + k-1  k 1and [S]   Kd
k1

Under these conditions, 1/Km = 1/Kd = affinity.

If k2 + k-1  k-1, then 1/Km underestimates the affinity 1/Kd

• Km equals to the dissociation constant of the ES complex if K 2 is much smaller than

K-1
• When this condition is met , Km is a measure of the strength of the ES complex: a
high Km indicates weak binding ; a low Km indicates strong binding

40
kcat/Km is a measure of catalytic efficiency
• Most enzymesare not fully saturated with substrate
• Under physiological conditions [S]/Km is 0.1 ̶ 1.0
• When [S] << Km the enzyme rate is very much less than Kcat as most sites are
unoccupied

Vo = k2[ES] [ES] = [E][S] / Km

= k2[E][S] / Km
= kcat / Km ([E][S])

• When [S] << Km the concentration of free enzyme ([E]) is nearly that of total
enzyme concentration ([Et])
Vo = kcat / Km ([Et][S])

• When [S] << Km, the enzymatic rate depends on kcat / K ratio, [Et] and [S]
– Under these conditions kcat / Km is the rate limiting ratio
• kcat / Km is a measure of the catalytic efficiency because it takes into account
both the rate of catalysis with a particular substrate (kcat) and the strength of
enzyme-substrate interaction (Km)
41
The Hill equation describes the behaviour of enzymes that exhibit
cooperative binding of substrate
-Some enzymes bind their substrates in a cooperative fashion analogous to the
binding of oxygen by hemoglobin
-Cooperative binding is encountered in multimeric enzymes that bind substrates
at multiple sites
-In enzymes that display positive cooperativity in binding to substrate, the
shape of the curve that relate vi to changes in [S] is sigmoidal
-Neither the Michaelis-Menten equation nor the Lineweaver-Burk plot can be
used to evaluate cooperative saturation kinetics

vi
Sigmoid saturation kinetics

[S]

42
 Kinetics of Enzyme-Catalyzed Reactions
Involving Two or More Substrates
• Usually, enzymes catalyze reactions involving two (or even more) substrates.
A +B → P + Q
• A bisubstrate reaction as above proceeds in one of the two ways
1. Both A and B are bound to the enzyme and then reaction occurs to give PQ:
E +A +B → AEB → PEQ → E + P + Q
– Reactions of this type are defined as sequential or single-displacement reactions. They can be either of two
distinct classes:
• random, where either A or B may bind to the enzyme first, followed by the other substrate, or
• ordered, where A, designated the leading substrate, must bind to E first before B can be bound.
• Both classes of single-displacement reactions are characterized by lines that intersect to the left of the
1/v axis in Lineweaver–Burk double reciprocal plots
2. The other general possibility is that one substrate, A, binds to the enzyme and reacts with it to yield a
chemically modified form of the enzyme (E) plus the product, P. The second substrate, B, then reacts
with E, regenerating E and forming the other product, Q.

– Reactions that fit this model are called ping-pong or double-displacement reactions. Two distinctive
features of this mechanism are the obligatory formation of a modified enzyme intermediate, E, and the pattern
of parallel lines obtained in double-reciprocal plots

43
 Single-displacement bisubstrate
mechanism. Double-reciprocal plots of
the rates observed with different fixed
concentrations of one substrate (B
here) are graphed versus a series of
concentrations of A. Note that, in these
Lineweaver–Burk plots for
singledisplacement bisubstrate
mechanisms, the lines intersect to the
left of the 1/v axis.

Double-displacement (pingpong)
bisubstrate mechanisms are characterized
by Lineweaver–Burk plots of parallel lines
when double-reciprocal plots of the rates
observed with different fixed concentrations of
the second substrate, B, are graphed versus a
series of concentrations of A.

44
Random, Single-Displacement Reactions
• In this type of sequential reaction, all possible binary enzyme : substrate complexes (AE, EB,
QE, EP) are formed rapidly and reversibly when the enzyme is added to a reaction mixture
containing A, B, P, and Q:

• The rate-limiting step is the reaction AEB→QEP

Random, single-
displacement bisubstrate
mechanism where A does
not
affect B binding, and vice
versa. Note that the lines
intersect at the 1/[A] axis. (If
[B] were varied in an
experiment with several fixed
concentrations of A, the lines
would intersect
at the 1/[B] axis in a 1/v
• If A has no influence on the binding constant for B (and vice versa); that is, the mechanism is purely
versus 1/[B] plot.)
random. Then, the lines in a Lineweaver–Burk plot intersect at the 1/[A] axis

45
• Creatine kinase acts by a random, single-displacement mechanism

46
Ordered, Single-Displacement Reactions
• In this case, the leading substrate, A (also called the obligatory or compulsory substrate), must bind first.
– Then the second substrate, B, binds.
– Strictly speaking, B cannot bind to free enzyme in the absence of A.
• Reaction between A and B occurs in the ternary complex, and is usually followed by an ordered release
of the products of the reaction, P and Q.

Or

• Note that A and Q are competitive for binding to the free enzyme, E, but not A and B (or Q and B).

47
NAD-dependent dehydrogenases show ordered single-displacement mechanisms
• Nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenases behave
according to the kinetic pattern just described. A general reaction of these
dehydrogenases is

• The leading substrate (A) is nicotinamide adenine dinucleotide (NAD), and NAD and
NADH (product Q) compete for a common site on E. A specific example is offered by
alcohol dehydrogenase (ADH)

48
Double-Displacement (Ping-Pong) Reactions
• Reactions conforming to this kinetic pattern are characterized by the fact
that the product of the enzyme’s reaction with A (called P in the following
schemes) is released prior to reaction of the enzyme with the second
substrate, B.
• As a result of this process, the enzyme, E, is converted to a modified form,
E, which then reacts with B to give the second product, Q, and regenerate
the unmodified enzyme form, E

49
These schemes predict that A and Q compete for the free enzyme form, E, while
B and P compete for the modified enzyme form, E. A and Q do not bind to E, nor
do B and P combine with E.

50
Aminotransferases Show Double-Displacement Catalytic Mechanisms
• One class of enzymes that follow a ping-pong-type mechanism are
aminotransferases (previously known as transaminases).
• These enzymes catalyze the transfer of an amino group from an amino acid to an –
keto acid.
• The products are a new amino acid and the keto acid corresponding to the carbon
skeleton of the amino donor

Glutamate : aspartate aminotransferase, an

enzyme conforming to a double-displacement
bisubstrate mechanism. Glutamate : aspartate
aminotransferase is a pyridoxal phosphate–
dependent enzyme. The pyridoxal serves as
the ͟NH2 acceptor from glutamate to form
pyridoxamine. Pyridoxamine is then the amino
donor to oxaloacetate to form asparate and
regenerate the pyridoxal coenzyme form. (The
pyridoxamine : enzyme is the E form.)

51
*********

52
Mechanisms of enzyme
action

53
Stabilization of transition states
• In all chemical reactions, the reacting atoms or molecules pass
through a state that is intermediate in structure between the
reactant(s) and the product(s).

• The catalytic role of an enzyme is to reduce the energy barrier

between substrate and transition state.
– This is accomplished through the formation of an enzyme–substrate
complex (ES)
– This complex is converted to product by passing through a transition
state, EX‡
– As shown, the energy of EX‡ is clearly lower than X‡

54
Enzymes catalyze reactions by lowering the activation energy.
Here the free energy of activation for (a) the uncatalyzed
reaction, Gu‡, is larger than that for (b) the enzyme–catalyzed
reaction, Ge‡.

55
• The energy barrier for the uncatalyzed reaction is of course the difference in
energies of the S and X‡ states.
• The energy barrier to be surmounted in the enzyme-catalyzed reaction,
assuming that E is saturated with S, is the energy difference between ES and
EX‡
• Reaction rate acceleration by an enzyme means simply that the energy barrier
between ES and EX‡ is less than the energy barrier between S and X‡
• In terms of the free energies of activation, ∆Ge‡˂ ∆Gu‡
– The enzyme must stabilize the transition-state complex, EX‡, more than it
stabilizes the substrate complex, ES.
– Put another way, enzymes are “designed” by nature to bind the transition-
state structure more tightly than the substrate (or the product).
• The dissociation constant for the enzyme-substrate complex is

and the corresponding dissociation constant for the transition-state complex is

Enzyme catalysis requires that KT ˂ KS

56
• There are a limited number of catalytic mechanisms or factors that
contribute to the remarkable performance of enzymes.
• These include the following:
– Entropy loss in ES formation
– Destabilization of ES due to strain, desolvation, or electrostatic effects
– Covalent catalysis
– General acid or base catalysis
– Metal ion catalysis
– Proximity and orientation
• Any or all of these mechanisms may contribute to the net rate acceleration
of an enzyme-catalyzed reaction relative to the uncatalyzed reaction
• The formation of the enzyme-substrate (ES) complex makes all these
mechanisms possible

57
The Binding Energy of ES Is Crucial to Catalysis
• The favorable interactions between the substrate and amino acid residues
on the enzyme account for the intrinsic binding energy, Gb.
• The intrinsic binding energy ensures the favorable formation of the ES
complex, but, if uncompensated, it makes the activation energy for the
enzyme catalyzed reaction unnecessarily large and wastes some of the
catalytic power of the enzyme
The intrinsic binding energy of the
enzyme-substrate (ES) complex (Gb) is
compensated to some extent by entropy
loss due to the binding of E and S (TS)
and by destabilization of ES (Gd) by
strain, distortion, desolvation, and similar
effects. If Gb were not compensated by
TS and Gd, the formation of ES would
follow the dashed line.

58
No distabilization of ES. Distabilization of ES.
No catalysis Facilitates catalysis
(a) Catalysis does not occur if the ES complex and the transition
state
for the reaction are stabilized to equal extents. (b) Catalysis will
occur if the transition state is stabilized to a greater extent than
the ES complex (right). Entropy loss and destabilization of the ES
complex Gd ensure that this will be the case.
59
• Because the enzymatic reaction rate is determined by the difference in
energies between ES and EX‡, the smaller this difference, the faster the
enzyme-catalyzed reaction.
• Tight binding of the substrate deepens the energy well of the ES complex
and actually lowers the rate of the reaction.

60
Entropy Loss and Destabilization of the ES Complex
• raising the energy of ES will increase the enzyme-catalyzed reaction rate.
• This is accomplished in two ways:
– (a) loss of entropy due to the binding of S to E, and
– (b) destabilization of ES by strain, distortion, desolvation, or other similar effects .

Transition-State Analogs Bind Very Tightly to the Active Site

• Transition-state analogs, are stable molecules that are chemically and
structurally similar to the transition state
• Such molecules should bind more strongly than a substrate and more
strongly than competitive inhibitors that bear no significant similarity to the
transition state.

61
(i) Catalysis by proximity
-molecules must come within bonding distance
-Higher concentrations increase the rate of encounters resulting in increased rate of
reaction
-When enzyme binds substrate molecules it creates a region of high local
concentration
-also orients substrate molecules spatially in a position ideal for
catalysis
(ii) Acid-base catalysis
-Many biochemical reactions involve the formation of unstable charged intermediates
that tend to breakdown rapidly to their constituent reactant species – thus
impeding the reaction
-Charged species can often be stabilized by the transfer of protons to and from
substrate or intermediate to form a species that breaks down more readily to
products than to reactants
-Acid –base catalysis can be either specific or general
.Catalysis that uses only the H+ and OH─ present in water, is referred
to as specific acid or specific base catalysis
. General acid or general base catalysis refers to transfers
mediated by other classes of molecules

62
-Ionizable functional groups of aminoacyl side chains ± prosthetic groups
contribute to catalysis by acting as acids or bases
-Proton transfers are the most common biochemical reactions

(iii) Catalysis by strain

-In enzymes that catalyze lytic reactions
-enzymes bind substrate in a way unfavourable for the bond that will
undergo cleavage
-The strain stretches the bond - weakens it - vulnerable to cleavage
(iv) Covalent catalysis
- Involves the formation of a transient covalent bond between the enzyme and one or
more substrates
- Modified enzyme becomes a reactant (transient modification)
-new pathway that is metabolically favourable – faster
- Enzyme returns to its original unmodified form
- Covalent catalysis is common among enzymes that catalyze group transfer
reactions
- Residues on the enzyme that participate in covalent catalysis generally
are cysteine or serine and occasionally histidine
- Covalent catalysis often follows a ‘ping –pong’ mechanism
-first substrate is bound and its product released prior to the
binding of the second substrate 63
V -Metal ions
Can participate in catalysis using several mechanisms
-may facilitate formation of nucleophiles such as hydroxide by direct
coordination eg zinc ions in carbonic acid anhydrase
-may serve as electrophiles, stabilizing a negative charge on an
intermediate
- may serve as bridges between enzyme and substrate increasing the
binding energy . This is the case in almost all enzymes that utilize ATP
as substrate
through the above mechanisms metal ions can play a role in
-general acid-base catalysis
-covalent catalysis
-approximation of reactants
-induction of strain in the enzyme or substrate

64
Enzyme Kinetics - Inhibition

65
Types of Inhibition
• Competitive Inhibition
• Noncompetitive Inhibition
• Uncompetitive Inhibition
• Irreversible Inhibition

66
 1. Competitive Inhibition
Enzyme
S
I

•In competitive inhibition, the inhibitor competes with the substrate for the
same binding site
•In competitive inhibition, the inhibitor binds only to the free enzyme, not to
the ES complex

67
 Competitive inhibition reaction mechanism
• Both the substrate and inhibitor compete for
binding to the same form of the enzyme:
free form
 ESI complex is not formed

• The inhibition is most noticeable at low [S]

but can be overcome at sufficiently high [S]
 Vmax remains unaffected
• Attaining Vmax requires higher [S] in the presence of competitive
inhibitor
 Apparent Km is increased

• Competitive inhibitors are especially attractive as clinical modulators of enzyme activity

because they offer two routes for the reversal of enzyme inhibition
1.like all kinds of reversible inhibitors, a decreasing concentration of the inhibitor reverses
the equilibrium
2.since substrate and competitive inhibitors both bind at the same site, raising [S], while
holding [I] constant, provides the second route for reversal of competitive inhibition

68
 Competitive Inhibition

Vmax - No change
Km INCREASES - indicates a direct interaction
of the inhibitor in the active site
69
 General Michaelis-Menten Equation
In the presence of a competitive inhibitor, the Michaelis-Menten equation becomes

where

• This form of the Michaelis-Menten equation can be used to understand how

each type of inhibitor affects the reaction rate curve
• In competitive inhibition, only the apparent Km is affected (Km,app> Km),
• The Vmax remains unchanged by the presence of the inhibitor.

70
 Competitive inhibitors alter the apparent Km, not the Vmax
.

- Inhibitor
Vmax
Reaction Rate

+ Inhibitor
Vmax
Vmax,app = Vmax
2
Km,app > Km

Km Km,app
[Substrate]
71
 The Lineweaver-Burk plot is diagnostic for competitive inhibition

1 = Km,app 1
+ 1 Increasing [I]
v Vmax [S] Vmax

Km,app
1 Slope =
Vmax
v

1
Vmax

-1 1
Km,app
[S] 72
Relating the Michaelis-Menten equation, the v vs. [S] plot, and the physical picture of
competitive inhibition

Inhibitor competes
with
substrate, decreasing S
its apparent affinity:
.

Km,app > Km
I .

- Inhibitor
Vmax

Reaction Rate
+ Inhibitor
E+S ES E+P Vmax
+ 2
I Formation
FormationofofEI
EI
Km,app > Km
Vmax,app = Vmax
complex
complexshifts
shiftsreaction
reaction
totothe
theleft:
left:KKm,app > Kmm
m,app > K
EI Km Km,app
73
[Substrate]
 Example - Competitive Inhibition
NH2
Sulfanilamide is a competitive inhibitor
of p-aminobenzoic acid.
folic acid Sulfanilamides (also known as sulfa
drugs, discovered in the 1930s)
were the first effective systemic
COOH
p-aminobenzoic acid antibacterial agents.
NH2
Because we do not make folic acid,
sulfanilamides do not affect human
cells.

SO2 NH2
sulfanilamide

74
Practical case: Methanol poisoning

A wealthy visitor is taken to the emergency

room, where he is diagnosed with
methanol poisoning. You are contacted
by a 3rd year medical student and asked
what to do? How would you suggest
treating this patient?

Methanol (CH3OH) is metabolized to

formaldehyde and formic acid by alcohol
dehydrogenase. You advise the third year
student to get the patient very drunk. Since
ethanol (CH3CH2OH) competes with methanol
for the same binding site on alcohol
dehydrogenase, it slows the metabolism of
methanol, allowing the toxic metabolites to be
disposed of before they build up to dangerous
levels. By the way, the patient was very
grateful and decided to leave all their worldly
possessions to the hospital. Unfortunately,
after being released from the hospital, he
75
went to the casinos and lost everything he
had.
2. Noncompetitive Inhibition
.

I I • the inhibitor does not

S interfere with substrate
Enzyme S Enzyme binding (and vice versa)

S
I I
S
Enzyme Enzyme

E+S ES E+P
+ + In noncompetitive
I I inhibition, the
inhibitor binds
enzyme
EI + S ESI regardless of
whether the
substrate is
bound 76
.

Noncompetitive inhibitors decrease the V max,app, but don’t affect the Km

Vmax - Inhibitor
1 = Km 1 1
Reaction Rate

+ Increasing [I]
v Vmax,app [S] Vmax,app

Vmax,app
1 Slope =
Km
1
V
+ Inhibitor v Vmax,app
2 max
1
V
2 max,app 1
Vmax,app
-1
Km
1
[S]
Km [Substrate] The Lineweaver-Burk plot is diagnostic for
Km,app noncompetitive inhibition
Vmax,app < Vmax
Km,app = Km

77
 Why does Km,app = Km for noncompetitive inhibition?

E+S ES E+P
+ +
I I The inhibitor binds equally well to free
enzyme and the ES complex, so it doesn’t
alter apparent affinity of the enzyme for
the substrate
EI + S ESI

78
 Relating the Michaelis-Menten equation, the v vs. [S] plot, and the physical picture of
noncompetitive inhibition
.

I I
.

Vmax - Inhibitor
S

Reaction Rate
Enzyme S Enzyme
Vmax,app
Inhibitor doesn’t interfere
with substrate binding, 1
V
+ Inhibitor
2 max
Km,app = Km Vmax,app < Vmax
1
V
2 max,app
S
I I Km,app = Km
S
Enzyme Enzyme Km Km,app
[Substrate]

E + Formation
S ES
of EI
E+P
+ complex shifts+ reaction
Even at high
I to the left: K I substrate
>K levels,
m,app m
inhibitor still binds,
[E]t < [ES]

EI + S ESI Vmax,app < Vmax

79
Noncompetitive inhibitors
decrease the apparent Vmax,
but do not alter the Km of the
reaction

80
 Example of noncompetitive inhibition: fructose 1,6-bisphosphatase inhibition by AMP

O O
- -
O P O- O P O-
fructose 1,6- O O
diphosphate H2C CH2
fructose 6-
O phosphate
H HO
H OH
OH H
Pi
fructose 1,6-
diphosphate
fructose 1,6- fructose 1,6- fructose 1,6-
bisphosphatase bisphosphatase bisphosphatase

E E.S E+P
AMP AMP AMP AMP

AMP AMP
O O
-O -O
P O- P O-
fructose 1,6- O O
diphosphate H C O CH2
2

H HO
H OH
OH H
fructose 1,6-
diphosphate
fructose 1,6- fructose 1,6-
bisphosphatase bisphosphatase

E.I E.S.I
• Fructose 1,6-bisphosphatase is a key regulatory enzyme in the gluconeogenesis
pathway. High amounts of AMP signal that ATP levels are low and gluconeogenesis
should be shut down while glycolysis is turned on.
• High AMP levels inhibit fructose 1,6-bisphosphatase (shutting down
gluconeogenesis) and activate phosphofructokinase-1 (turning on glycolysis).
Regulation of fructose 1,6-bisphosphatase and phosphofructokinase-1 by AMP
prevents a futile cycle in which glucose is simultaneously synthesized and broken
down. 81
Uncompetitive Inhibition
Enzyme.

Enzyme

S
S
In uncompetitive inhibition, the inhibitor binds
Enzyme only to the ES complex
I I

Enzyme

I S

E+S ES E+P Mechanism of uncompetitive inhibition

+ In uncompetitive inhibition, the inhibitor
binds only to the ES complex, it does
I not bind to the free enzyme

ESI 82
 .

Uncompetitive inhibitors decrease both the Vmax,app and the Km,app

Reaction Rate Vmax - Inhibitor Vmax,app < Vmax

Km,app < Km
Vmax,app
1 + Inhibitor
Notice that at low substrate
V
1
2 max
concentrations, uncompetitive
V
2 max,app inhibitors have little effect on the
reaction rate because the lower
Km,app of the enzyme offsets the
Km,app Km [Substrate]
decreased Vmax,app

1 = Km,app 1 1
+
v Vmax,app [S] Vmax,app 1 Increasing [I]

=
Km 1
+
1 v
Vmax [S] Vmax,app
Km The Lineweaver-Burk plot is diagnostic for
Slope =
Vmax
uncompetitive inhibition

1
Vmax,app

-1 1
Km,app
[S]
83
Uncompetitive inhibitors decrease both the V max,app and the Km,app of the enzyme

E+S ES E+P Notice that uncompetitive inhibitors

+ don’t bind to the free enzyme, so
there is no EI complex in the
I reaction mechanism

ESI

84
 Relating the Michaelis-Menten equation, the v vs.
[S] plot, and the physical picture of uncompetitive
inhibition
Enzyme.

Enzyme

Vmax - Inhibitor

Reaction Rate
S
S
Enzyme
Vmax,app
I I 1
V
2 max
+ Inhibitor
Inhibitor 1
V Vmax,app < Vmax
increases Enzyme
2 max,app

the amount of Km,app< Km

enzyme bound
to substrate I S Km,app Km
.

[Substrate]
Km,app < Km

E+S ES E+P Uncompetitive inhibitors decrease the

+ Even at high
Formation of EI
apparent Km of the enzyme and decrease
substrate levels,
I
complex shiftsinhibitor
reactionbinds,
the Vmax of the reaction

to the left: Km,app

[E] ><K[ES]
m
t
Vmax,app < Vmax 85
ESI
 Example of uncompetitive inhibition:
alkaline phosphatase inhibition by
.
phenylalanine
Alkaline Alkaline Alkaline
phosphatase phosphatase phosphatase

O O
O P O- -
O P O-
O O-
O
O-
P
O
O

-
-

Phe Phe

Alakaline
Phosphatase

O
O P O-
Phe O-
86
 Irreversible Inhibition
In irreversible inhibition, the
inhibitor binds to the enzyme
Enzyme irreversibly through formation of
a covalent bond with the enzyme
S , permanently inactivating the
enzyme
O I

87
Irreversible Inhibition - Reaction
Mechanism
E+S ES E+P
+
In irreversible inhibition, the inhibitor
I permanently inactivates the enzyme.
The net effect is to remove enzyme from
the reaction.
EI Vmax decreases
No effect on Km

88
The Michaelis-Menten plot for an irreversible inhibitor looks like noncompetitive
.

inhibition

Vmax - Inhibitor
Reaction Rate
Vmax,app
1
V
+ Inhibitor
2 max
1
V
2 max,app

Km [Substrate]
Km,app
Vmax,app < Vmax
Km,app = Km

89
Irreversible inhibition is distinguished from noncompetitive inhibition by plotting V max vs [E]t
.

Enzyme is inactivated

or t
ibi
r until all of the
ito

Inh
it or irreversible inhibitor is
hib

ib
Inh

ble
used up
- In
Vmax

ib le ive

rs i
e rs etit
ev omp

ve
R
+ nc
rr e
No +I

[E]t

[E]t < [I] [E]t > [I]

[E]t = [I]
Irreversible inhibitors decrease Vmax,app, but leave the
apparent Km unchanged. Irreversible inhibitors differ
from other types of inhibitors because they covalently
modify the enzyme. This results in the permanent
inhibition of the enzyme activity. 90
 Examples of Irreversible Inhibitors
• diisopropylphosphofluoridate
– prototype for the nerve gas sarin
– permanently inactivates serine proteases by forming a covalent bond
with the active site serine

91
 Penicillin is a suicide inhibitor
R
O Penicillin
C
S CH3
H
H N

HC CH3

N COO-
C
H
O Strained
peptide bond R
O
glycopeptide C
glycopeptide H S CH3
transpeptidase transpeptidase
H N

HC CH3

N COO-
Ser OH Ser O C
H
H
O

Glycopeptide transpeptidase catalyzes the formation of cross-links

between amino acids in the cell walls of bacteria. This enzyme also
catalyzes the reverse reaction, the hydrolysis of peptide bonds. During
the course of hydrolyzing the strained peptide bond in penicillin, the
enzyme activates the inhibitor (penicillin), which then covalently
modifies an active site serine in the enzyme. In effect, the enzyme
“commits suicide” by hydrolyzing the strained peptide bond in penicillin.
92
Summary-Enzyme Inhibition
• Competitive Inhibitor
– Binds to substrate binding site
– Competes with substrate
– The affinity of the substrate appears to be decreased when
inhibitor is present (Km,app >Km)
• Noncompetitive inhibitor
– Binds to allosteric site
– Does not compete with the substrate for binding to the
enzyme
– The maximum velocity appears to be decreased in the
presence of the inhibitor (Vmax,app <Vmax)

93
• Uncompetitive Inhibitor
– Binds to the enzyme only after the substrate has bound
– The affinity of the substrate appears to be increased and the
maximum velocity appears to be decreased when inhibitor is
present (Km,app <Km, Vmax,app <Vmax),
• Irreversible Inhibitor
– Covalently modifies and permanently inactivates the enzyme

94