Enzymes

• Virtually all reactions in the body are mediated by

enzymes
• Protein catalysts
• Increase the rate of reactions without being
changed in the overall process
• Direct all metabolic events
• Have the suffix “-ase” attached to the substrate of
the reaction ; Glucosidase, Urease
• Increase the velocity of a chemical reaction and are
not consumed during the reaction
• Contain a special pocket or cleft called Active site
• Active site; participates in substrate binding and
catalysis
CLASSIFICATION OF
ENZYMES
• Activation energy; Minimum amount of energy required
for reactants to form products in a chemical reaction
• Enzymes; Lower the activation energy for reactions
• Enzyme-catalyzed reactions are one thousand to one
hundred millions times faster than uncatalyzed reactions
 Effect of Substrate Concentration on
Reaction Velocity

• Vmax = Maximal velocity

• Km = Michaelis constant
 Km; Michaelis constant

• Characteristic of an enzyme and

its particular substrate
• Reflects the affinity of the enzyme
for that substrate
• Km= 1⁄2 Vmax
• Km; Substrate concentration which
permits the enzyme to achieve half
Vmax
• Km does not vary with Enzyme
concentration
 Small Km
• A numerically small (low) Km reflects a high affinity of
the enzyme for substrate
• Because a low concentration of substrate is needed to half
saturate the enzyme—that is, to reach 1⁄2Vmax
 Large Km
• A numerically large (high) Km reflects a low affinity of
enzyme for substrate
• Because a high concentration of substrate is needed to half
saturate the enzyme
Enzymes
• Highly specific
• Catalyze only one type of chemical reaction
 Some enzymes require molecules
Other than proteins for Enzymatic activity
• If Nonprotein moiety is a metal ion, such as Zn2+ or
Fe2+, it is called as COFACTOR
• If Nonprotein moiety is a small organic molecule, it is
termed COENZYME
• Coenzymes commonly are derived from Vitamins
Alcohol Dehydrogenase
Cofactor; Zinc
Coenzyme; NAD
Vitamin B @ Coenzymes
• Enzyme activity can be regulated,
• Increased or decreased, so that
• The rate of product formation responds to cellular need
• The reaction velocity increases with temperature
until a peak velocity is reached
• This increase is the result of ;
Increased number of molecules having sufficient
energy to pass over the energy barrier and form
the products of the reaction
• Decrease of velocity with higher temperature
• Further elevation of the temperature causes a
decrease in reaction velocity as a result of
Temperature induced denaturation of the enzyme
 pH affects reaction velocity

• Pepsin, a digestive enzyme in Stomach, is maximally

active at pH 2
• Other enzymes, designed to work at neutral pH, are
denatured by such an acidic environment
• Extremes of pH;
• Can also lead to denaturation of the enzyme
• Because the structure of the catalytically active protein
molecule depends on;
• Ionic character of Amino acid side chains
INHIBITION OF ENZYME ACTIVITY
• Any substance that can decrease the velocity of an
enzyme catalyzed reaction is called an inhibitor
• Inhibitors can be reversible or irreversible

• Reversible inhibitors; Bind to enzymes through

Noncovalent bonds
• Irreversible inhibitors ; Bind to enzymes through
Covalent bonds
 INHIBITION OF ENZYME ACTIVITY

• Lead forms covalent bonds with

the sulfhydryl side chain of
Cysteine in proteins
• Ferrochelatase and ALA
dehdratase are;
• Involved in Hemoglobin synthesis,
• Irreversibly inhibited by Pb
 Competitive Enzyme Inhibition

• Occurs when Inhibitor binds reversibly to the same site

that the substrate would normally occupy
• Competes with the substrate for that site
• Effect of a competitive inhibitor is reversed by increasing
Substrate concentration ( Basis of therapeutic usage of
Ethanol in Methanol poisoning )
 Competitive enzyme inhibitors
‘’Statin drugs’’
• Competitively inhibits the rate limiting step in Cholesterol
synthesis
• Reaction is catalyzed by Hydroxymethylglutaryl–CoA
reductase (HMG-CoA reductase)
 Statin Drugs
• Structural analogs of
natural substrate for
HMG CoA reductase
• Compete effectively to
inhibit the enzyme
• So, they inhibit
Cholesterol synthesis
• Lowers blood Cholesterol
levels
 ALCOHOL METABOLISM IN LIVER

• Ethanol is first converted to Acetaldehyde by Alcohol

dehydrogenase
• Acetaldehyde is produced
 Acetaldehyde is highly reactive and TOXIC
• Can covalently bind to proteins , lipids, and nucleic acids
• Forms Acetaldehyde adducts, which, in turn,
• Can disrupt the structure and function of these
macromolecules
• Some people exhibit facial flushing after consuming only
modest amounts of ethanol, due to acetaldehyde
accumulation
 Disulfiram

• Used in the treatment of Chronic alcoholism

• Inhibits Aldehyde dehydrogenase
• Results;
• Increase in Acetaldehyde
• Flushing, Tachycardia, Hyperventilation, and Nausea
Alcohol Metabolism
• Formic acid is toxic to optic nerves
• If Unterated; Blindness, Acidose, Coma and Death
 Methanol poisoning is treated by Ethanol
• Ethanol is a competitive inhibitor of Alcohol
dehydrogenase
• It’s affinity is 10-20 times greater than that of Methanol
• Ethanol slows the rate of methanol’s convertion to
Formaldehyde and Formic acid
 Enzyme Inhibitors as Drugs

• Penicillin and Amoxicillin, act by

• Inhibiting enzymes involved in bacterial cell wall synthesis
 Enzyme Inhibitors as Drugs

• Aspirin; inhibits Prostaglandin and Thromboxane synthesis

 REGULATION OF ENZYME ACTIVITY
• Some enzymes with specialized regulatory functions;
• Respond to;
• Allosteric effectors and/or Covalent modification or
• They show altered rates of enzyme synthesis or
degradation when physiologic conditions are changed
Regulation of Allosteric enzymes
• Allo steri = Different site
• Allosteric enzymes;

• Regulated by molecules called effectors that bind

Noncovalently at a site other than the active site
 Allosteric Enzymes
• Almost always composed of multiple subunits
• Regulatory (Allosteric) site that binds the effector
is distinct from the substrate binding site
• Effectors that inhibit enzyme activity are termed
Negative effectors,
• Whereas those that increase enzyme activity are
called Positive effectors
ALLOSTERIC REGULATION
 Regulation of Enzymes by
Covalent Modification
• Many enzymes are regulated by Covalent modification

• Most often by Addition or Removal of Phosphate groups

from specific Serine, Threonine, or Tyrosine residues of the
enzyme
• Phosphorylation reactions are catalyzed by a family
of enzymes called Protein kinases that use ATP as
Phosphate donor
• Phosphate groups are cleaved from phosphorylated
enzymes by the action of Phosphatases
Response of Enzyme to Phosphorylation
• Depending on the specific enzyme;
• Phosphorylated form may be more or less active
than Unphosphorylated enzyme
 Induction and Repression of Enzyme Synthesis
• Cells can also regulate the amount of enzyme present by;
• Altering the rate of enzyme degradation or , more
typically, the rate of enzyme synthesis
• Increase (Induction) or decrease (Repression) of Enzyme
synthesis ;
• Leads to alteration in total population of active sites
• Affects the amount of enzyme
Induction and Repression of Enzyme Synthesis

• Elevated levels of insulin as a result of high blood

glucose levels cause ;
• Increase in synthesis of key enzymes involved in
Glucose metabolism
Induction and Repression of Enzyme Synthesis

• Elevated levels of insulin as a result of high blood glucose

levels cause ;
• Increase in synthesis of key enzymes involved in Glucose
metabolism
• Alterations in enzyme levels as a result of induction or
repression of protein synthesis are slow (hours to days),
compared with allosterically or covalently regulated
changes in enzyme activity, which occur in seconds to
minutes
ENZYMES IN CLINICAL DIAGNOSIS
• Plasma is the fluid, noncellular part of blood
• A relatively small group of plasma enzymes are
actively secreted into blood by certain cell types
• Liver secretes Zymogens (Inactive precursors) of
Enzymes involved in Blood Coagulation
 ENZYMES IN CLINICAL DIAGNOSIS

• A large number of plasma enzyme species are released

from cells during normal cell turnover
• These enzymes almost always function intracellularly and
have no physiologic use in the plasma
 ENZYMES IN CLINICAL DIAGNOSIS
• In healthy individuals;
• The levels of these enzymes are fairly constant
• Represent a steady state in which
• The rate of release from damaged cells into plasma is
balanced by an equal rate of removal from plasma
• Increased plasma levels of these enzymes may indicate
tissue damage
 Alteration of Plasma Enzyme Levels
In Disease States
• Many diseases that cause tissue damage result in
• Increased release of intracellular enzymes into plasma
• Activities of many of these enzymes are routinely
determined for diagnostic purposes in diseases of ;
• Heart
• Liver
• Skeletal muscle, and other tissues
 Alteration of Plasma Enzyme Levels
In Disease States
• The level of specific enzyme activity in the plasma
frequently correlates with the extent of tissue damage
• Determining the degree of elevation of a particular
enzyme activity in the plasma is
• Often useful in evaluating the prognosis for the patient
 Plasma Enzymes as Diagnostic Tools
• Some enzymes show relatively high activity in only one or
a few tissues
• The presence of increased levels of these enzymes in
plasma thus reflects damage to the corresponding tissue

• Alanine aminotransferase ([ALT]) is abundant in Liver

• Appearance of elevated levels of ALT in plasma signals
possible damage to hepatic tissue
• Measurement of ALT is part of the liver function test panel
 Isoenzymes
‘’Isozymes’’
• Catalyze the same reaction
• Do not necessarily have the same physical properties
because of genetically determined differences in amino
acid sequence
• For this reason;
• May contain different numbers of charged amino acids and
• May be separated from each other by electrophoresis
 Isoenzymes

• Different organs commonly contain characteristic

proportions of different isoenzymes
• The pattern of isoenzymes found in the plasma may,
therefore, serve as a means of identifying the site of tissue
damage
 Isoenzymes and Heart diseases

• Plasma levels of Creatine Kinase (CK) are commonly

determined in the diagnosis of Myocardial Infarction (MI)
• They are particularly useful when the electrocardiogram is
difficult to interpret such as when there have been
previous episodes of heart disease
 Diagnosis of Myocardial Infarction
• Measurement of blood levels of proteins with Cardiac
tissue specificity is used
• Myocardial muscle is the only tissue that contains more
than 5% of the total CK activity as CK (MB) isoenzyme
• Increase of CK-MB in plasma is virtually specific for MI
• CK-MB is used as Cardiac marker for Myocardial tissue
damage
• Following an acute MI
• CK (MB) appears approximately 4–6 hours following onset
of chest pain
• Reaches a peak of activity at approximately 12-24 hours
• Returns to baseline after 48–72 hours
• In combination with the clinical presentation and
characteristic changes in Electrocardiogram, are currently
considered the “Gold standard” in the diagnosis of a MI