Enzymes
- mediate all reactions in the body
- protein catalysts that increase the rate of reactions without themselves being changed in the overall
process
- channel reactants (substrates) into useful pathways
- provide speed, specificity, and regulatory control (regulating the rate of metabolic pathways) to
reactions in the body
- may be modified during their participation in this reaction sequence, they return to their original form at
the end
- share some properties with chemical catalysts and differ in several ways
Shared Properties
- enzymes are neither consumed nor produced during the course of a reaction
- enzymes do not cause reactions to take place
- expedite reactions that would ordinarily proceed but at a much slower rate
- do not alter the equilibrium constants of reactions they catalyze
Differences Between Enzymes and Chemical Catalysts
a. Enzymes
- are proteins
- are highly specific for the reactions they catalyze and produce only the expected
products from the given reactants
- high specificity toward 1 substrate (some enzymes have broader specificity using more
than 1 substrate)
- function within a moderate pH and temperature range
- higher reaction rates
- capacity for regulation
Measures of Enzyme Activity
1. Unit of Enzyme Activity
- amount of an enzyme that catalyzes the transformation of 1 micromole of substrate per minute
under optimal conditions of measurement
2. Specific Activity
- number of units of enzyme activity per milligram of total protein present
3. Turnover Number (Kcat)
- number of substrate molecules metabolized per enzyme molecule per unit time
NOMENCLATURE
A. Recommended Name
- most commonly used name
- suffix “-ase” attached to the
- substrate of the reaction (ex: glucosidase, urease, sucrase)
- description of the action performed (ex: lactate dehydrogenase, adenylate cyclase)
- some enzymes retain their original trivial name (no hint of the associated enzymatic action)
(ex: trypsin, pepsin)
B. Systematic Name (by the International Union of Biochemistry and Molecular Biology)
- 6 major classes with subgroups
- suffix “-ase” attached to complete the name (ex: D-glyceraldehyde 3-phosphate:NAD oxidoreductase)
PROPERTIES of ENZYMES
- increase the velocity of a chemical reaction
1. Ribozymes
- RNAs with catalytic activity
- catalyze cleavage and synthesis of phosphodiester bonds
A. Enzyme Binding Sites
- bind substrates through interactions with the amino acid residues of the enzyme
1. Spatial Geometry
- required for all the interactions between the substrate and the enzyme
- makes each enzyme selective for its substrates
- ensures that only specific products are formed
- overlap in the active catalytic site of the enzyme
B. Active Catalytic Site
- region of the enzyme where the reaction occurs
Functional Groups
- within the catalytic site
- provided by
- coenzymes
- tightly bound metals
- amino acid residues of the enzyme (amino acid side chains create 3-dimensional
surface (special pocket or cleft) complimentary to the substrate)
- participate in catalysis
Catalytic Efficiency
- highly efficient
1. Catalytic Power of an Enzyme
- rate of the catalyzed reaction divided by the rate of the uncatalyzed reaction (106
-1014 x faster
than uncatalyzed reactions)
2. Turnover Number
- each enzyme molecule is capable to transform 100-1000 substrate molecules into product per
second
Specificity
- ability of an enzyme to select just one substrate and distinguish this substrate from a group of very
similar compounds
- highly specific
- interact to 1 or few substrates
- catalyze only 1 type of biochemical reaction
- the specificity and speed of enzyme-catalyzed reactions result from the unique sequence of specific
amino acids that form the three-dimensional structure of the enzyme
- the enzyme and its substrate(s) must have geometric, electronic, and stereospecific complementarity
Determined By
- functional groups of the substrate (or product)
- functional groups of the enzyme and its cofactors
- physical proximity of these various functional groups
Cofactors
- enzymes associate with nonprotein cofactors needed for enzymatic activity
1. Holoenzyme
- enzyme with its cofactor
- active
2. Apoenzyme
- protein portion of the holoenzyme
- does not show biologic activity in the absence of cofactor [enzyme without its cofactor(s)]
- inactive
3. Prosthetic Group
- tightly bound coenzyme that does dissociate from the enzyme
Two Classes
a. Metals
- eg: Mg2+
Zn2+
Fe++
b. Small Organic Molecules (Coenzymes)
- often vitamin derivatives
- eg: biotin
THF
NAD+
CoA
FAD
Vitamin Deficiencies
- can lead to cofactor deficiencies holoenzymes left unformed inability for
certain cellular reactions to occur phenotypic manifestations of
vitamin deficiency disease states
- ex: Vitamin B1 (Thiamine) Deficiency
thiamine pyrophosphate (TPP) deficiency pyruvate
dehydrogenase, -ketoglutarate dehydrogenase,
and transketolase remain in their inactive,
apoenzymatic forms cells' ability to produce
energy drastically reduced beriberi (neurologic
dysfunction, cardiac dysfunction, weight loss)
The ENZYME-CATALYZED REACTION
A. Catalytic Mechanisms
1. Acid-Base Catalysis
- occurs when partial proton transfer from an acid and/or partial proton abstraction by a base
lowers the free energy of a reaction’s transition state
- pH-dependent catalytic rates
- ex: RNase A
- has two catalytic His residues which act as general acid and general base
catalysts
2. Covalent Catalysis
- reversible formation of a covalent bond stabilization of the transition state of the reaction
through electron delocalization
- ex: Nucleophilic attack on the substrate by the enzyme to form a Schiff base intermediate
capable of stabilizing (lowering the free energy of) a developing negative charge
a. Common Nucleophiles
- negatively charged or contain unshared electron pairs
- imidazole and sulfhydryl groups
3. Metal Ion Catalysis
- metalloenzymes tightly bind catalytically essential transition metal ions
- metal-activated enzymes loosely bind metal ions such as Na+
, K+
, or Ca2+ that play a structural
role
a. Metal Ions
- orient substrates for reaction
- mediate oxidation-reduction reactions
- electrostatically stabilize or shield negative charges
b. Zn2+ Ion of Carbonic Anhydrase
- makes a bound water molecule more acidic increase the concentration of the
nucleophile OH-
4. Catalysis by Proximity and Orientation Effects
- enzymes lower the activation energies of the reactions they catalyze by bringing their
reactants into proximity
- by properly orienting them for reaction
- by using charged groups to stabilize the transition state (electrostatic catalysis)
- by freezing out the relative motions of the reactants and the enzyme’s catalytic groups
5. Catalysis by Preferential Binding of the Transition State
- enzyme’s preferential binding of the transition state lowers ΔG increases the rate of the
reaction (an unreactive compound that mimics the transition state may be an effective
enzyme inhibitor)
Three Basic Steps
1. Binding of Substrate
E + S ES
- enzyme binds the substrates of the reaction it catalyzes and brings them together at the right
orientation to react
2. Conversion of Bound Substrate to Bound Product
ES EP
- enzyme participates in the making and breaking of bonds required for product formation
3. Release of Product
EP E + P
- enzyme releases the products and returns to its original state once the reaction is completed
C. The Active Site
- cleft or crevice in the enzyme formed by one or more regions of the polypeptide chain
- cofactors and functional groups from the polypeptide chain participate in transforming the bound
substrate molecules into products
- substrate molecules bind to their substrate binding sites (substrate recognition sites)
- the three-dimensional arrangement of binding sites in a crevice of the enzyme allows the reacting
portions of the substrates to approach each other from the appropriate angles
- proximity of the bound substrate molecules and their precise orientation toward each other contribute to
the catalytic power of the enzyme
- active site also contains functional groups that directly participate in the reaction
- functional groups are donated by the polypeptide chain or by bound cofactors (metals or complex
organic molecules called coenzymes)
- as the substrate binds, it induces conformational changes in the enzyme that promote further interactions
between the substrate molecules and the enzyme functional groups
- ex: coenzyme might form a covalent intermediate with the substrate, or an amino acid side
chain might abstract a proton from the reacting substrate
- activated substrates and the enzyme form a transition state complex, an unstable high-energy complex
with a strained electronic configuration that is intermediate between substrate and product
- additional bonds with the enzyme stabilize the transition state complex and decrease the energy required for its formation
- free enzyme then binds another set of substrates and repeats the process
Substrate Binding Sites
1. Lock-and-Key Model for Substrate Binding
- substrate binding site contains amino acid residues arranged in a complementary three-
dimensional surface that “recognizes” the substrate and binds it through multiple
hydrophobic interactions, electrostatic interactions, or hydrogen bonds
- amino acid residues that bind the substrate can come from very different parts of the linear amino
acid sequence of the enzyme
“Induced Fit” Model for Substrate Binding
- as the substrate binds, enzymes undergo a conformational change (“induced fit”) that
repositions the side chains of the amino acids in the active site and increases the number
of binding interactions
- the function of the conformational change induced by substrate binding, the induced fit, is
usually to reposition functional groups in the active site in a way that promotes the
reaction, improves the binding site of a cosubstrate, or activates an adjacent subunit
through cooperativity
The Transition State Complex
1. Energy Changes Occurring During the Reaction
a. Free Energy of Activation
- energy barrier separating the reactants and the products
- energy difference between that of the reactants and a high-energy intermediate that
occurs during the formation of a product
- difference in energy between the substrate and the transition state complex
b. Transition State
- condition in which bonds in the substrate are maximally strained in some enzyme-
catalyzed reactions
- electronic configuration of the substrate becomes very strained and unstable as it
enters the transition state in other enzyme-catalyzed reactions
c. Transition State Theory
- the reactants of a reaction pass through a short-lived high-energy state that is
structurally intermediate to the reactants and products
- overall rate of the reaction is determined by the number of molecules acquiring the
activation energy (energized molecules) necessary to form the transition state
complex
- for molecules to react, they must contain sufficient energy to overcome the energy
barrier of the transition state (large activation energy slow rates of
uncatalyzed chemical reactions)
- the lower the free energy of activation more molecules have sufficient energy to pass
over the transition state faster rate of reaction
d. Alternate Reaction Pathway
i. Enzymes
- increase the rate of the reaction by decreasing the activation energy (provides
a pathway with a lower free energy of activation)
- does not change the free energies of the reactants or products
- does not change the equilibrium of the reaction
Chemistry of the Active Site
- complex molecular machine employing a diversity of chemical mechanisms to facilitate the
conversion of a substrate to a product
- factors responsible for the catalytic activity of the enzyme
a. Transition State Stabilization
- act as a flexible molecular template that binds the substrate in a geometry
structurally resembling the activated transition state of the molecule
- stabilized substrate transition state enzyme greatly increases the concentration of the
reactive intermediate that can be converted to product reaction accelerated
b. Other Factors
- active site provides catalytic groups
- enhance the probability that the transition state forms
- participate in general acid-base catalysis (amino acid residues provide or
accept protons)
- transient formation of covalent enzyme-substrate complex
SERINE PROTEASES
- widespread family of enzymes that have a common mechanism
Active Sites
a. Active-Site Ser
- identified through its inactivation by diisopropylphosphofluoridate
b. Active-Site His
- identified through affinity labelling with a chloromethylketone substrate analog
c. Active-Site Asp
- identified by X-ray crystallography
- form a hydrogen-bonded catalytic triad
1. Nonhomologous Serine Proteases
- have developed the same catalytic triad through convergent evolution
B. Catalysis by Serine Proteases
- multistep process
- nucleophilic attack on the scissile bond by Ser 195 (using the chymotrypsinogen numbering system)
tetrahedral intermediate that decomposes to an acyl-enzyme intermediate
- the replacement of the amine product with water is necessary for the formation of a second tetrahedral
intermediate which yields the carboxyl product and regenerated enzyme
- involving the Ser-His-Asp triad
- formation of the tetrahedral intermediates
3. Catalysis Through Binding of the Transition State
- in the oxyanion hole
C. Low-Barrier Hydrogen Bonds (LBHBs)
- proposed to play important roles in the stabilization of transition states
- strong hydrogen bonds
- isolated from other water molecules
- predicted bond energies 4 times those of normal hydrogen bonds in aqueous environments
FUNCTIONAL GROUPS in CATALYSIS
A. Functional Groups on Amino Acid Side Chains
- almost all of the polar amino acids participate directly in catalysis in one or more enzymes
1. Amino Acids in Covalent Catalysis
a. Serine
b. Cysteine
c. Lysine
d. Histidine
- has a pKa
that can donate and accept a proton at neutral pH
- often participates in acid-base catalysis
2. Amino Acids in Nucleophilic Catalysis
- most of the polar amino acid side chains are nucleophilic and participate in by stabilizing
more positively charged groups that develop during the reaction
a. Nucleophiles
- carry full or partial negative charges (like the oxygen atom in the serine -OH) or have a
nitrogen that can act as an electron-donating group by virtue of its two
unpaired electrons
b. Nucleophilic Groups
- carried out
- covalent catalysis
- acid-base catalysis
c. Electrophiles
- carry full or partial positive charges (e.g., peptide backbone -NH was used as an
electrophilic group in chymotrypsin)
Nucleophilic and Electrophilic Catalysis
- occur when the respective nucleophilic or electrophilic groups on the enzyme
stabilize substrate groups of the opposite polarity that develop during the
reaction
B. Coenzymes in Catalysis
1. Coenzymes
- complex nonprotein organic molecules
- participate in catalysis by providing functional groups
- usually (but not always) synthesized from vitamins
- most coenzymes are tightly bound to their enzymes and do not dissociate during the course of
the reaction
- most coenzymes, such as functional groups on the enzyme amino acids, are regenerated during
the course of the reaction
- involved in catalyzing a specific type of reaction for a class of substrates with certain structural
features
- very little activity in the absence of the enzyme and very little specificity
- the enzyme provides specificity, proximity, and orientation in the substrate
recognition site, as well as other functional groups for stabilization of the
transition state, acid-base catalysis, etc.
Most Vitamins
- function as coenzymes
- the symptoms of vitamin deficiencies reflect the
loss of specific enzyme activities dependent
on the coenzyme form of the vitamin
Drugs and Toxins
- that inhibit proteins required for coenzyme
synthesis (e.g., vitamin transport proteins or
biosynthetic enzymes) can cause the
symptoms of a vitamin deficiency
(functional deficiency)
Dietary Deficiency
- inadequate intake
Ethanol - “antivitamin” that decreases the cellular content of
almost every coenzyme
- inhibits the absorption of thiamine through the
intestinal mucosal cells
- acetaldehyde produced from ethanol oxidation
displaces pyridoxal phosphate from its
protein binding sites, thereby accelerating
its degradation
Two General Classes
a. Activation-Transfer Coenzymes
- usually participate directly in catalysis by forming a covalent bond with a portion of
the substrate (portion of the coenzyme that forms a covalent bond with the
substrate is its functional group)
- the tightly held substrate moiety is then activated for transfer, addition of water, or
some other reaction
- a separate portion of the coenzyme binds tightly to the enzyme
i. Thiamine Pyrophosphate
ia. Synthesis
- synthesized in human cells from the vitamin thiamine by the addition
of a pyrophosphate
ib. Pyrophosphate
- provides negatively charged oxygen atoms that chelate Mg++, which
then binds tightly to the enzyme
ic. Reactive Carbon Atom
- functional group
- extends into the active site
- with a dissociable proton
- forms a covalent bond with a substrate keto group while cleaving
the adjacent carbon-carbon bond
id. Function
- coenzyme in the decarboxylation of -keto acids such as pyruvate
and -ketoglutarate
- coenzyme in the utilization of pentose phosphates in the pentose
phosphate pathway
- thiamine deficiency
- oxidation of -keto acids is impaired
- dysfunction occurs in the central and peripheral nervous
system, the cardiovascular system, and other organs
For Your Eyes
Other Activation-Transfer Coenzymes
- also synthesized from vitamins
iia. Coenzyme A (CoA)
- synthesized from the vitamin pantothenate
- contains an adenosine 3’,5’-bisphosphate which binds reversibly,
but tightly, to a site on an enzyme
- sulfhydryl group at the other end of the molecule
- nucleophile that always attacks carbonyl groups and
forms acyl thioesters
iib. Biotin
- does not contain a phosphate group
- covalently bound to a lysine in carboxylases
- nitrogen atom
- covalently binds a CO2 group in an energy-requiring
- bound CO2 group is activated for addition to another
molecule
- functions only in carboxylation reactions in humans
iic. Pyridoxal Phosphate
- synthesized from pyridoxine (vitamin B6)
- aldehyde group
- reactive
- usually functions in enzyme-catalyzed reactions by forming
a covalent bond with the amino groups on amino
acids
- ring nitrogen
- positively charged
- withdraws electrons from a bond in the bound amino acid,
resulting in cleavage of that bond
- the enzyme participates by removing protons from the substrate and
by keeping the amino acid and the pyridoxal group in a single
plane to facilitate shuttling of electrons
Three Common Features of all Activation-Transfer Coenzymes
iiia. Specific Chemical Group
- involved in binding to the enzyme
iiib. Separate and Different Functional or Reactive Group
- participates directly in the catalysis of one type of reaction by forming
a covalent bond with the substrate
iiic. Dependence of the Enzyme
- for additional specificity of substrate and additional catalytic power
Oxidation-Reduction Coenzymes
- involved in oxidation-reduction reactions
- catalyzed by enzymes categorized as oxidoreductases
- follow the same principles as activation-transfer coenzymes
- do not form covalent bonds with the substrate
- each coenzyme has a unique functional group that accepts and donates electrons and
is specific for the form of electrons it transfers (e.g., hydride ions, hydrogen
atoms, oxygen)
- different portion of the coenzyme binds the enzyme
- not good catalysts without participation from amino acid side chains on the enzyme
Oxidation of a Compound
- loses electrons oxidized carbon has fewer H
atoms or gains an O atom
Reduction of a Compound
- gain of electrons gain of H, or loss of O
i.Nicotinamide Adenine Dinucleotide (NAD+
) and Flavin Adenine Dinucleotide
(FAD)
- can transfer electrons together with hydrogen
- unique roles in the generation of ATP from the oxidation of fuels
ii. Other Oxidation-Reduction Coenzymes
- work with metals to transfer single electrons to oxygen
iia. Vitamin E and Vitamin C (Ascorbic Acid)
- oxidation-reduction coenzymes that can act as antioxidants and
protect against oxygen-derived free radical injury
iii. Nicotinamide Adenine Dinucleotide (NAD+
)
iiia. Synthesis
- synthesized from the vitamin niacin (which forms the nicotinamide
ring), and from ATP (which contributes an AMP)
iiib. ADP Portion
- binds tightly to the enzyme
- causes conformational changes in the enzyme
iiic. Carbon on the Nicotinamide Ring Opposite the Positively Charged
Nitrogen
- accepts the hydride ion (hydrogen atom that has two electrons)
transferred from a specific carbon atom on the substrate
- H
+
from the substrate alcohol (-OH) group then dissociates, and a
keto group (C=O) is formed
- the enzyme contributes a histidine nitrogen that can bind the
dissociable proton on lactate, thereby making it easier for
to pull off the other hydrogen with both electrons
Oxidation of Lactate to Pyruvate
1. Lactate
- loses two electrons as a hydride ion, and a
proton (H+
) is released
2. NAD+
- accepts the hydride ion, is reduced to
NADH
- the carbon atom with the keto group is
now at a higher oxidation state
because both of the electrons in
bonds between carbon and oxygen
are counted as belonging to oxygen,
whereas the two electrons in the
C-H bond are shared equally
between carbon and hydrogen
C. Metal Ions in Catalysis
- have a positive charge
- contribute to the catalytic process by acting as electrophiles (electron-attracting groups)
- assist in binding of the substrate, or they stabilize developing anions in the reaction
- can also accept and donate electrons in oxidation-reduction reactions
1. Mg++ - role in the binding of the negatively charged phosphate groups of thiamine pyrophosphate
to anionic or basic amino acids in the enzyme
- phosphate groups of ATP
- usually bound to enzymes through Mg++ chelation
2. Metals of Some Enzymes
- bind anionic substrates or intermediates of the reaction to alter their charge distribution,
thereby contributing to catalytic power
a. Alcohol Dehydrogenase
- transfers electrons from ethanol to NAD+
- in the active site of alcohol dehydrogenase, an activated serine pulls a proton off the
ethanol -OH group new electronic configuration (negative charge on the
oxygen that is stabilized by zinc) allows the transfer of a hydride ion to
In humans, most of ingested ethanol is oxidized to acetaldehyde
in the liver by alcohol dehydrogenase (ADH):
Ethanol + NAD+
Acetaldehyde + NADH + H+
ADH - active as a dimer
- active site containing zinc present in each subunit
Humans have at least seven genes that encode isozymes of
ADH, each with a slightly different range of
specificities for the alcohols it oxidizes
Acetaldehyde
- highly reactive, toxic, and immunogenic
- responsible for much of the liver injury associated
with chronic alcoholism
A patient was admitted to the hospital after intravenous
thiamine was initiated at a dose of 100 mg/day
(compared with an RDA of 1.4 mg/day). His congestive
heart failure was believed to be the result, in part, of the
cardiomyopathy (heart muscle dysfunction) of acute
thiamine deficiency known as beriberi heart disease.
This nutritional cardiac disorder and the peripheral
nerve dysfunction usually respond to thiamine
replacement. However, an alcoholic cardiomyopathy
can also occur in well-nourished patients with adequate
thiamine levels. Exactly how ethanol, or its toxic
metabolite acetaldehyde, causes alcoholic
cardiomyopathy in the absence of thiamine deficiency is
unknown.
At low concentrations of ethanol, liver alcohol
dehydrogenase is the major route of ethanol
oxidation to acetaldehyde, a highly toxic
chemical. Acetaldehyde not only damages the
liver, it can enter the blood and potentially
damage the heart and other tissues. At low
ethanol intakes, much of the acetaldehyde
produced is safely oxidized to acetate in the
liver by acetaldehyde dehydrogenases.
Noncatalytic Roles of Cofactors
1. Noncatalytic structural role in certain enzymes
2. Bind different regions of the enzyme together to form the tertiary structure
3. As substrates that are cleaved during the reaction
INHIBITION of ENZYME ACTIVITY
1. Inhibitor
- any substance that can diminish the velocity of an enzyme-catalyzed reaction
a. Reversible Inhibitors
- bind to enzymes through noncovalent bonds
- dilution of enzyme-inhibitor complex dissociation of the reversibly-bound inhibitor
recovery of enzyme activity
b. Irreversible Inhibition
- inhibited enzyme does not regain activity upon dilution of the enzyme-inhibitor
- inhibitors form covalent bonds with specific groups of the enzyme
Competitive Inhibition
- inhibitor binds reversibly to the same site that the substrate would normally occupy ( competes with the
substrate for that site) therefore is usually a close structural analog of the substrate
1. Effect on Vmax
- no effect on Vmax
- effect of competitive inhibitor is reversed by increasing [S]
- sufficiently high [S] reaction velocity reaches Vmax
2. Effect on Km
- when the substrate concentration is increased to a sufficiently high level, the substrate binding
sites are occupied by substrate, and inhibitor molecules cannot bind
competitive inhibitors, therefore, increase the apparent Km of the enzyme (Km app ) because
they raise the concentration of substrate necessary to saturate the enzyme [reduces the
apparent affinity of the enzyme for its substrate (increases Km)]
- more substrate is needed to achieve 1⁄2Vmax
3. Effect on Lineweaver-Burke Plot
- plots of the inhibited and uninhibited reactions intersect on the y axis at 1/Vmax (Vmax is
unchanged)
- inhibited and uninhibited reactions show different x-axis intercepts
apparent Km is increased in the presence of competitive inhibitor
Some of a patient’s problems have arisen from product
inhibition of liver alcohol dehydrogenase by NADH. As
ethanol is oxidized in liver cells, NAD+
is reduced to NADH
and the NADH/NAD+
ratio rises. NADH is an inhibitor of
alcohol dehydrogenase, competitive with respect to NAD+
,
so the increased NADH/NAD+
ratio slows the rate of
ethanol oxidation and ethanol clearance from the blood.
NADH is also a product inhibitor of enzymes in the
pathway that oxidizes fatty acids. Consequently, these fatty
acids accumulate in the liver, eventually contributing to the
alcoholic fatty liver.
Noncompetitive (Mixed) Inhibition
- inhibitor and substrate bind at different sites on the enzyme
- binds to both free and substrate-bound enzyme
- may interfere with both substrate binding and catalysis
- when only Vmax is affected, the inhibition is said to be noncompetitive
- noncompetitive inhibition cannot be overcome by increasing [S] decrease the Vmax
- noncompetitive inhibitors do not interfere with the binding of substrate to enzyme enzyme
shows the same Km with or without a noncompetitive inhibitor
3. Effect on the Lineweaver-Burke Plot
- Vmax decreases in the presence of a noncompetitive inhibitor
- Km is unchanged
4. Examples of Noncompetitive Inhibitors
- some inhibitors act by forming covalent bonds with specific groups of enzymes
a. Lead (Pb) Poisoning
- Pb forms covalent bonds with sulfhydryl side chains of cysteine in proteins
- enzymes sensitive to inhibition by Pb
- ferrochetalase
- -aminolevulinate dehydrase
b. Other Examples
- certain insecticides irreversible binding at the catalytic site of acetylcholinesterase
neurotoxicity
Uncompetitive Inhibition
- inhibitors bind only to the ES at a site distinct from the active site (allosteric site) and apparently distorts
the active site
- binds only to the enzyme-substrate complex
- increases the apparent Km and decreases Vmax
1. Ki
- dissociation constant for the ES-inhibitor complex
D. Irreversible Competitive Inhibitors
- bind covalently or so tight to the active site of the enzyme
1. Affinity Labels
- substrate analogues that possess a highly reactive group reacts covalently with an amino acid
residue (not necessarily involved in catalysis) permanently block active site of the
enzyme from the substrate
2. Mechanism-Based/Suicide Inhibitors
- substrate analogues that are transformed by the enzyme
- mimic or participate in an intermediate step of the catalytic reaction
- ex: many drugs
toxins
a. Covalent Inhibitors
- form covalent or extremely tight bonds with functional groups in the active catalytic
site
- functional groups
- activated by their interactions with other amino acid residues, and
are therefore far more likely to be targeted by drugs and toxins
than amino acid residues outside the active site
i. Diisopropyl Phosphofluoridate (DFP, or Diisopropylfluorophosphate)
- lethal compound
- organophosphorus compound
- served as a prototype for the development of the nerve gas Sarin and other
organophosphorus toxins, such as the insecticides malathion and
parathion
- exerts its toxic effect by forming a covalent intermediate (inhibition is
essentially irreversible) in the active site of acetylcholinesterase,
thereby preventing the enzyme from degrading the neurotransmitter
acetylcholine
- activity can only be recovered as new enzyme is synthesized
Acetylcholinesterase
- cleaves the neurotransmitter acetylcholine to acetate and
choline in the postsynaptic terminal, thereby
terminating the transmission of the neural signal
Malathion
- metabolized in the liver to a toxic derivative (malaoxon)
that binds to the active site serine in
acetylcholinesterase and other enzymes
acetylcholine accumulates and overstimulates
the autonomic nervous system (involuntary nervous
system, including heart, blood vessels, glands)
vomiting, abdominal cramps, salivation, and
sweating
Acetylcholine
- also a neurotransmitter for the somatic
motor nervous system
involuntary muscle twitching
(muscle fasciculations)
- also inhibits many other enzymes that use serine for hydrolytic cleavage, but
the inhibition is not as lethal
A patient survived his malathion intoxication because he
had ingested only a small amount of the chemical,
vomited shortly after the agent was ingested, and was
rapidly treated in the emergency room. Lethal doses of
oral malathion are estimated at 1 g/kg of body weight for
humans. Emergency room physicians used a drug (oxime)
to reactivate the acetylcholinesterase before the aged
complex formed. They also used intravenous atropine, an
anticholinergic (antimuscarinic) agent, to antagonize
the action of the excessive amounts of acetylcholine
accumulating in cholinergic receptors throughout his body.
After several days of intravenous therapy, the signs and
symptoms of acetylcholine excess abated, and therapy was
slowly withdrawn. The patient made an uneventful recovery.
For Your Eyes Only
Once ingested, the liver converts malathion to the
toxic reactive compound, malaoxon, by replacing
the sulfur with an oxygen. Malaoxon then binds to
the active site of acetylcholinesterase and reacts to
form the covalent intermediate. Unlike the
complex formed between diisopropylfluorophosphate
and acetylcholinesterase, this initial acylenzyme
intermediate is reversible. However, with time, the
enzyme-inhibitor complex “ages” (dealkylation of
the inhibitor and enzyme modification) to form an
irreversible complex.
ii. Aspirin (Acetylsalicylic Acid)
- exerts its effect through the covalent acetylation of an active site serine in the
enzyme prostaglandin endoperoxide synthase (cycloxygenase)
- resembles a portion of the prostaglandin precursor that is a physiologic
substrate for the enzyme
Transition State Analogs and Compounds that Resemble Intermediate Stages of the
Reaction
- resemble the electronic and three-dimensional surface of the natural substrate
- more potent inhibitors than are substrate analogs
- extremely potent
- specific inhibitors of enzymes
- bind so much more tightly to the enzyme than do substrates or products
- drugs cannot be designed that precisely mimic the transition state because of its highly
unstable structure
i. Drug Developed as a Transition State Analog
- would be highly specific for the enzyme it is designed to inhibit
- highly unstable when not bound to the enzyme and would have great
difficulty making it from the digestive tract or injection site to the site
of action
ii. Approaches in Drug Design to Deal with the Instability Problem
- designing drugs that are almost transition state analogs but have a stable
modification
- designing a prodrug that is converted to a transition state analog at the site of
action
- using the transition state analog to design a complementary antibody
Substrates undergo progressive changes in their overall
electrostatic structure during the formation of a transition
state complex, and effective drugs often resemble an
intermediate stage of the reaction more closely than they
resemble the substrate (medical literature often refers to
such compounds as substrate analogs, even though they
bind more tightly than substrates)
Abzymes (Catalytic Antibodies)
- made as antibodies against analogs of the transition state complex
- have an arrangement of amino acid side chains in their variable regions that is
similar to the active site of the enzyme in the transition state
- can act as artificial enzymes
- ex: abzymes against analogs of the transition state complex of cocaine
esterase (enzyme that degrades cocaine in the body)
- have esterase activity
- monthly injections of the abzyme drug can be used to
rapidly destroy cocaine in the blood, thereby
decreasing the dependence of addicted individuals
c. Substrate Analogues
- closely resemble the transition state of the natural substrate
- binds to the active site of the enzyme without covalent modification
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