Enzyme Inhibitors as Drugs
1. Penicillin
- transition state analog that binds very tightly to glycopeptidyl transferase
a. Glycopeptidyl Transferase
- enzyme required by bacteria for synthesis of the cell wall
- catalyzes a partial reaction with penicillin that covalently attaches penicillin to its own
active site serine
- reaction is favoured by the strong resemblance between the peptide bond in the
-lactam ring of penicillin and the transition state complex of the natural
transpeptidation reaction
2. Allopurinol
- drug used to treat gout
- decreases urate production by inhibiting xanthine oxidase
- converted to a transition state analog
a. Xanthine Oxidase
- oxidation of hypoxanthine to xanthine and xanthine to uric acid (urate) in the
pathway for degradation of purines
- contains a molybdenum-sulfide (Mo-S) complex that binds the substrates and
transfers the electrons required for the oxidation reactions
- oxidizes the drug allopurinol to oxypurinol
b. Oxypurinol
- binds very tightly to a molybdenum-sulfide complex in the active site
3. Angiotensin-Converting Enzyme (ACE) Inhibitors
- captopril
- lisinopril
- enalapril
- inhibit extracellular reactions
- block enzyme that cleaves angiotensin I to form angiotensin II (potent vasoconstrictor)
lowering of BP
4. Statin Drugs
a. Antihyperlipidemic Agents
- atorvastatin
- simvastatin
structural analogs of the natural substrate
- competitively inhibits the 1st committed step in cholesterol synthesis catalyzed by
hydroxymethylglutaryl CoA reductase (HMG CoA reductase) inhibition of
de novo cholesterol synthesis lowering plasma cholesterol levels
F. Heavy Metals
1. Examples
a. Mercury (Hg)
b. Lead (Pb)
c. Aluminum (Al)
d. Iron (Fe)
2. Heavy Metals
- relatively nonspecific for the enzymes they inhibit
- binds to so many enzymes, often at reactive sulfhydryl groups in the active site that it
has been difficult to determine which of the inhibited enzymes is responsible for
toxicity
b. Lead - inhibits through replacing the normal functional metal in an enzyme
- its developmental and neurologic toxicity may be caused by its ability to replace Ca++
in two regulatory proteins important in the central nervous system and other
tissues, Ca++
-calmodulin and protein kinase C
3. Heavy Metal Toxicity
- caused by tight binding of a metal to a functional group in an enzyme
REGULATION of ENZYME ACTIVITY
A. Regulation by Substrate and Product Concentration
1. Factors Affecting Reaction Velocity
a. Substrate Concentration
i. Maximal Velocity (Vmax)
- rate/velocity of a reaction (v)
= number of substrate molecules converted to product per unit time
- micromoles product formed / min
- rate of enzyme-catalyzed reaction increases with substrate concentration
until a Vmax is reached
- high substrate concentration saturation of all binding sites on the
enzyme levelling off of the reaction
ii. Hyperbolic Shape of Velocity vs. [Substrate]
- most enzymes show
- Michaelis-Menten kinetics
- plot of reaction velocity (Vo) against substrate concentration ([S])
- allosteric enzymes
- show sigmoidal curve
iii. Michaelis-Menten Model of Enzyme Kinetics
- provide a quantitative way of describing the dependence of enzyme rate on
substrate concentration
- applies to a simple reaction in which the enzyme and substrate form an
enzyme-substrate complex (ES) that can dissociate back to the free
enzyme and substrate
iiia. Reaction Model
K1 K3
E + S ES E + P
K2
S - substrate
E - enzyme
ES - enzyme-substrate complex
K1, K2, K3 - rate constants
iiib. Michaelis-Menten Equation
- relates the initial velocity (vo) to the concentration of substrate [S] and
the two parameters Km and Vmax
Vo = Vmax [S]
Km + [S]
Vo - initial reaction velocity
- proportionate to the concentration of
enzyme-substrate complexes [ES]
Vmax - maximal velocity that can be achieved at
an infinite concentration of
substrate
- saturation kinetics
- velocity cannot increase any
further once the enzyme is
saturated with substrate
= K3 [ET]
The use of Vmax in the medical literature to describe the
maximal rate at which a certain amount of tissue converts
substrate to product can be confusing. The best way to
describe an increase in enzyme activity in a tissue is to say
that the maximal capacity of the tissue has increased.
Kcat - describe the speed at which an enzyme
can catalyse a reaction under
conditions of saturating substrate
concentration
- turnover number of the enzyme, has the
units of min-1 (micromoles of
product formed per minute divided
by the micromoles of active site).
Km - Michaelis-Menten constant
= (K2 + K3)/K1
- concentration of substrate at which vo
equals 1⁄2Vmax
- Km of an enzyme for a substrate is related
to the dissociation constant, Kd
(rate of substrate release divided by
the rate of substrate binding)
[S] - substrate concentration
Important Conclusions about Michaelis-Menten Kinetics
iiici. Characteristics of Michaelis Constant (Km)
- reflects the affinity of the enzyme for the substrate
- numerically equal to [substrate] at which the reaction
velocity is equal to 1⁄2Vmax
- does not vary with [enzyme]
iiicia. Small Km
- reflects high affinity of the enzyme for the
substrate (low [substrate] is needed to half-
saturate the enzyme)
- velocity of an enzyme is most sensitive to changes
in substrate concentration over a
concentration range below its Km
- at substrate concentrations less than 1/10th of the
Km, a doubling of substrate concentration
nearly doubles the velocity of the reaction
iiicib. Large Km
- reflects low affinity of the enzyme for the
substrate (high [substrate] is needed to half-
- at substrate concentrations 10 times the Km,
doubling the substrate concentration has
little effect on the velocity
- the higher the Km , the higher is the substrate
concentration required to reach 1⁄2Vmax
v = k[A]1
= k[A]
iiicii. Relationship of Velocity to [Enzyme]
- the rate of reaction is directly proportional to the [enzyme] at
all substrate concentrations
iiiciii. Order of Reaction
iiiciiia. First Order
- rate (velocity) is proportional to the reactant
- [S] < Km reaction velocity is proportional to
[substrate]
v = k[A]
iiiciiib. Zero Order
- [S] > Km and equal to Vmax
- reaction velocity is constant and is independent of
[reactant] ([substrate])
v = k[A]0
= k
iiiciiic. Second Order
- rate is proportional to the product of the
concentrations of the reactants
v = k[A]2
iiiciv. Michaelis-Menten Kinetic Theory of Enzyme Action
a. Effect of [Enzyme] on Reaction Velocity
- if [substrate] is constant reaction velocity is
proportional to [enzyme]
b. Effect of [Substrate] on Reaction Velocity
- if [substrate] is low reaction is 1st order with
respect to substrate
iiid. Lineweaver-Burke Plot (Double-Reciprocal Plot)
- used to calculate Km and Vmax
- determine the mechanism of action of enzyme inhibitors
1. Equation
1 = 1 + Km . 1
Vo Vmax Vmax [S]
2. Intercept on the x axis = -1/Km
3. Intercept on the y axis = 1/Vmax
Other Linear Transforms
1. Eadie-Hofstee Transform
v = Vmax - Km . v/[S]
2. Hanes-Woolf Transform
[S] = 1.[S] + Km
v Vmax Vmax
iv. Velocity and Enzyme Concentration
- reaction rate is directly proportional to the enzyme concentration
- if the amount of enzyme is doubled, the amount of product produced per
minute is also doubled whether the condition is at low or at saturating
concentrations of substrate
v. Multisubstrate Reactions
- most enzymes have more than one substrate, and the substrate binding sites
overlap in the catalytic (active) site
- the sequence of substrate binding and product release affect the rate equation
an apparent value of Km (Km app) depends on the concentration of
cosubstrate or product present
b. Temperature
- most human enzymes function optimally at a temperature of approximately 37oC
i. Increase of Velocity with Temperature
- increased number of molecules having sufficient energy to pass over the
energy barrier reaction velocity increases with temperature until a
peak velocity is reached
- maximum activity for most human enzymes occurs near 37oC because
denaturation (loss of secondary and tertiary structure) occurs at
higher temperatures
ii. Decrease of Velocity with Temperature
- further temperature elevation temperature-induced enzyme denaturation
decreased reaction velocity
c. pH
i. Effect of pH on Ionization of Active Site
- the shape of the curve in the acid region usually reflects the ionization of
specific functional groups in the active site (or in the substrate) by the
increase of pH, and the more general formation of hydrogen bonds
important for the overall conformation of the enzyme
- the loss of activity on the basic side usually reflects the inappropriate
ionization of amino acid residues in the enzyme
ii. Effect of pH on Enzyme Denaturation
- extreme pH enzyme denaturation
pH Changes
- alters the following
iva. Ionization state of the substrate or of the enzyme-binding site
for substrate or cofactor
ivb. Ionization state of the catalytic site on the enzyme
ivc. Conformation and catalytic activity of protein molecules change
Reversible Inhibition within the Active Site
a. Competitive Inhibition
b. Noncompetitive and Uncompetitive Inhibition
c. Simple Product Inhibition in Metabolic Pathways
- all products are reversible inhibitors of the enzymes that produce them and may be
competitive, noncompetitive, or uncompetitive relative to a particular substrate
i. Simple Product Inhibition
- a decrease in the rate of an enzyme caused by the accumulation of its own
product
- plays an important role in metabolic pathways
- it prevents one enzyme in a sequence of reactions from generating a product
faster than it can be used by the next enzyme in that sequence
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