ENZYME BIOCHEMISTRY

Compiled by…

Inyang, BASSEY atte

What are enzymes, and how do they
work?
• Enzymes are biological catalysts
• First “isolation” of an enzyme in 1833
• Ethanol added to aqueous extract of malt
• Yielded heat-labile precipitate that was utilized to hydrolyze starch to
soluble sugar; precipitate now known as amylase
• 1878 - Kühne coined term enzyme - means “in yeast”
• 1898 - Duclaux proposed all enzymes should have suffix “ase”
What are enzymes?
• Biological catalysts

• Involved in almost all chemical reactions that maintain homeostasis in

humans and animals
• Composed of proteins

• Except ribozymes (RNA based enzymes)

What are enzymes, and how do they
work?
• Enzymes have molecular weights of several thousand to several
million, yet catalyze transformations on molecules as small as carbon
dioxide and nitrogen
• Function by lowering transition-state energies and energetic
intermediates and by raising the ground-state energy
• Many different hypotheses proposed for how enzymes catalyze
reactions
• Common link of hypotheses: enzyme-catalyzed reaction always
initiated by the formation of an enzyme-substrate (or ES) complex in
a small cavity called the active site
ENZYMES
THE MOST IMPORTANT
PROTEINS IN YOUR BODY
IF asked whether a reaction is
possible…
THE answer is always YES!
ANYTHING, as we know…
IS POSSIBLE!!
An Enzyme…
• Brings substrates together in space and
time
• Lowers the free Energy of activation
• Stabilizes the hi energy intermediate
• Is not consumed in the reaction
An ENZYME has…
Active site contains amino acid residues and cofactors that are
responsible specificity and rate acceleration
 ENZYME
NOMENCLATURE
Classification of Enzymes
Enzyme classification
• Based on common name e.g. Alcohol dehydrogenase

• Currently enzymes are grouped into six functional classes by the

International Union of Biochemists (I.U.B.)

• Each enzyme is described by a sequence of one of four numbers

preceded by ‘EC’. The first number broadly classifies the enzyme
based on its mechanism.
Enzyme classification
• Based on composition
• Simple Enzymes – consist wholly of proteins
• Complex enzymes (Holoenzymes)

- Holoenzymes consist of apoenzyme + coenzyme or prosthetic group

(protein component) (non-protein component)
Enzyme classification………….

• Many coenzymes and prosthetic groups are derivatives of vitamins

• Non protein component of an enzyme may be as simple as a metal

ion OR as complex as a small non protein organic molecule

• Metalloenzymes : require a metal in high affinity in their composition

• Metal-activated enzymes : bind with low affinity to metal ion which is
required for its activity
Role of co-enzymes
• Transport chemical groups from one reactant to another eg
FAD,NAD,Pyridoxal phosphate

• * Assignment: Match coenzymes with the chemical groups they

transport
• Write a short note on Co-enzymes as second substrates
Enzyme relative to substrate type
• Two types of specificity(selectivity): (1) Specificity of binding and (2)
specificity of reaction
• Enzymes are highly reaction specific e.g. ADH always catalyze
oxidation reaction
• They are not always highly substrate specific e.g. ADH attacks a
number of different alcohols viz from methanol to butanol

• They have a broad range of substrate specificity e.g. Hexokinase

Enzymes relative to substrate………..
• Enzymes generally exhibit optical isomer(particular stearic
configuration)specificity

• Except racemases which show an exception to the rule

• Enzymes that attack D sugars will not attack L sugars (same for amino
acids)
 Enzyme relative to substrate
• A particular substrate may be attacked by a number of
different enzymes to produce the same product

• Enzymes sharing above characteristics are known as

Isozymes e.g LDH,CK
Enzyme -Substrate Interactions

• Induced fit hypothesis

• Lock and key hypothesis
• Concept of flexible active site
1958 - Induced-fit hypothesis proposed by
Koshland:
When a substrate begins to bind to an enzyme,
interactions induce a conformational change in the
enzyme
Results in a change of the enzyme from a low
catalytic form to a high catalytic form
Induced-fit hypothesis requires a flexible active site
Induced fit Model
• Relatively weak initial interaction between enzyme and
substrate
• Catalytic sites come in close proximity with substrate bonds
to be altered due to above
• Binding of E + S
• Generation of transition state complexes and reaction
products via one or more mechanisms of catalysis
Mechanisms of Catalysis

• Catalysis by Bond Strain

• Catalysis by Proximity and orientation
• Catalysis involving Donors(Acids) and Acceptors (Bases)
• Covalent catalysis
1894 - Lock-and-key hypothesis - Fischer
proposed enzyme is the lock into which the
substrate (the key) fits
Does not rationalize certain observed
phenomena:
Compounds having less bulky substituents
often fail to be substrates
Some compounds with more bulky substituents
bind more tightly
Some enzymes that catalyze reactions between
two substrates do not bind one substrate until
the other one is bound
Concept of flexible active site stated earlier by
Pauling (1946):
Hypothesized that an enzyme is a flexible template
that is most complementary to substrates at the
transition state rather than at the ground state
Therefore, the substrate does not bind most
effectively in the ES complex
As reaction proceeds, enzyme conforms better to
the transition-state structure
Transition-state stabilization results in rate
enhancement
• Only a dozen or so amino acid residues may make up
the active site
• Only two or three may be involved directly in substrate
binding and/or catalysis
EC 1. Oxidoreductases catalyse oxidation/reduction reactions.
• EC 2. Transferases transfer a functional group (e.g. a methyl
or phosphate group).
• EC 3. Hydrolases catalyse the hydrolysis of various bonds.
• EC 4. Lyases cleave various bonds by means other than
hydrolysis and oxidation.
• EC 5. Isomerases catalyse isomerisation changes within a
single molecule.
• EC 6. Ligases join two molecules with covalent bonds.
(The full nomenclature can be browsed at
http://www.chem.qmul.ac.uk/iubmb/enzyme/.)
Like all proteins, enzymes are made as linear chains of amino
acids that fold to produce
a three-dimensional product. Each unique amino acid sequence
produces a specific structure,
which has unique properties. Enzymes can be denatured – that
is, unfolded and inactivated
– by heating or by chemical denaturants, which disrupt the
three-dimensional structure of the
protein; denaturation may be reversible or irreversible.
How to name enzymes
FIRST NAME of an enzyme
IS the name of the SUBSTRATE
LAST NAME of an enzyme
Is what you did to the substrate
Competitive inhibition
• Inhibitor is similar to the substrate
• The inhibitor is competing for the active
site
• Affinity I decreased
• Km increases
• Vmax remains the same ( just add more
substrate)
• reversible
Noncompetitive inhibition
• NOT similar to the substrate
• Does NOT bind active site
• Binds to regulatory site
• Turns off the enzyme
• Km remains the same
• Vmax decreases
• irreversible
What makes a reaction favourable
and spontaneous
Factors affecting Enzyme Activity
• Concentration of chemicals involved in reaction
• Rate constants characteristic of the reaction

• A → B
• The rate of this reaction is expressed algebraically as either a decrease
in the concentration of reactant A:
• –[A] = k[B]
• or an increase in the concentration of product B:
• [B] = k[A]
 At equilibrium the rate (v) of the forward reaction
(A → B) is, by definition, equal to that of the reverse or
back reaction (B → A), a relationship which is
algebraically symbolized as:
vforward = vreverse

where, for the forward reaction:

vforward = k+1[A]

and for the reverse reaction:

vreverse = k–1[B]

In the above equations, k+1 and k–1 represent rate

constants for the forward and reverse reactions,
respectively.
Effects of temperature on a
reaction
Reactions,rates and equilibra
• Reaction rate
- Determined by number of molecules of reactant(s) converted into
products in a specified time

- Dependent on the concentration of the chemicals involved in the

process AND rate constants characteristic of the reaction
Reactions,rates and equilibra……..
• In the rxn in which A is converted to B,
(A → B)
Rxn rate is either a ↓ in the conc. of reactant A, −[A] = k[B] OR an ↑ in
The conc. of product B, [B] = k[A]
Rate constant for forward rxn is k+1, and for the reverse rxn k−1
Reactions,rates and equilibra……..
• At equilibrium, the rate of forward rxn = rate of reverse rxn

Equilibrium constant,,

Keq is equal to the ratio of product and reactant concentrations AND

also equal to the ration of the rate constants of the reaction
Reactions, rates and equilibria……..
• Catalysts speed up both forward and reverse rxns proportionately

• Although the magnitude of the rate constants of the forward and reverse
rxns is ↑ed;

• the ratio of the rate constants remains the same in the presence or absence
of enzymes

• Enzymes have no effect on the equilibrium constant of the reactions they

catalyze
Reactions, rates and equilibria……..
•
E + S ↔ ES ↔ ES* ↔ EP ↔ E + P

ES complex is formed when an enzyme and substrate meet;

This passes to (ES*), then to an enzyme product complex (EP), AND
finally there is dissociation to yield product and free enzyme.
Michaelis- menten equation
Michaelis- menten equation
• Describes the kinetics of enzyme
catalyzed reactions
• A quantitative description of the
relationship between;
V1 = rate of an enzyme-catalyzed
reaction,
[S] = the concentration of the
substrate,
(Vmax) = maximum reaction rate, AND
(Km) = Michaelis- Menten constant
Lineweaver burk equation and plot
• Introduced by biochemists Hans Lineweaver and Dean Burk

• Allows for analysis of enzyme kinetics based on the double reciprocal

plot of the Michaelis-Menten equation

• Averts the problem associated with dealing with curvilinear plots of

enzyme catalyzed reactions
Michaelis- menten equation and plot
• Has the same form as the equation
for a rectangular hyperbola

• graphical analysis of reaction rate (v)

versus substrate concentration [S]
produces a hyperbolic rate plot

• If the Michaelis–Menten plot is

extrapolated to infinitely high
substrate concentrations, the
extrapolated rate is equal to Vmax
Key features of Michaelis- menten plot
• Key features are marked by points A,B
and C
• Point C = rate of the reaction is essentially
equal to Vmax, AND the difference in rate
at nearby concentrations of substrate is
almost negligible;
-Rxn is occurring at zero order kinetics as it
is solely dependent on the amount of
enzyme .i.e rxn rate is independent of
substrate concentration
• Almost all the enzyme molecules are
bound to substrate
Key features of Michaelis- menten plot
• At points A and B, the lower reaction velocities, only a small amount
of enzyme molecules are bound to the substrate at any moment

• At substrate concentration denoted by point B,

- exactly half the enzyme molecules are in an ES complex;
- The rxn rate is exactly one half of Vmax
- The substrate concentration at this point is called Km
Key features of Michaelis- menten plot
• At points A and B, the lower reaction
velocities, only a small amount of
enzyme molecules are bound to the
substrate at any moment

• At substrate concentration denoted by

point B,
- exactly half the enzyme molecules are in
an ES complex;
- The rxn rate is exactly one half of Vmax
- The substrate concentration at this point
is called Km
Key features of Michaelis- menten plot
• At substrate concentration near
point A, the rate appears to be
directly proportional to substrate
concentration

• Reaction rate is said to be first

order
Key features of Michaelis- Menten plot
• What is Km?

• The substrate concentration at

which the rate of the enzyme
catalyzed reaction is exactly one
half of the maximum rxn
velocity, Vmax
Lineweaver burk equation and plot
• Introduced by biochemists Hans Lineweaver and Dean Burk

• Allows for analysis of enzyme kinetics based on the double reciprocal

plot of the Michaelis-Menten equation

• Averts the problem associated with dealing with curvilinear plots of

enzyme catalyzed reactions
Lineweaver-burk equation and plot
Application of Lineweaver- burk equation
• Useful in analysis of enzyme inhibition especially in the pharmaceutical
industry in which clinical drug therapy is based on inhibition of enzymes
e.g;
- Use of methotrexate in cancer chemotherapy to semi-selectively inhibit
DNA synthesis of malignant cells
- Inhibition of prostaglandins by aspirin in the treatment of aches and pains
- Inhibition of folic acid synthesis which is required for metabolism and
growth of disease causing bacteria
- Analysis of enzyme inhibition mediated by poisons (e.g cyanide,PCB,CO)
ENZYME INHIBITORS
• irreversible enzyme inhibitors
• reversible enzyme inhibitors
Irreversible enzyme inhibitors
• cause an inactivating, covalent modification of enzyme structure

• ↓se the concentration of active enzyme thus ↓ing maximum

possible ES complex;

• ↓ed enzyme concentration leads to ↓ed reaction rates

• e.g Cyanide (CN–) which covalently binds the ferric (Fe3+) iron required
for function of mitochondrial cytochrome oxidase
Classes of reversible enzyme inhibitors
• Competitive inhibitor

• Noncompetitive inhibitor

• Uncompetitive inhibitor
Simplified cases: Competitive inhibition
• 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
Simplified cases: Competitive inhibition
• 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

Examples of competitive inhibitors

2,3-biphosphoglycerate
• Inhibits its own formation by inhibiting biphosphoglycerate mutase
 Metabolic regulation by product inhibition
Examples of competitive
inhibitors

• Malonate vs succinate
Enzyme: succinate
dehydrogenase
• Krebs and his colleagues
used malonate to
investigate the TCA cycle
Examples of competitive
inhibitors

Sulphonamides: widely used in medicine to limit bacterial growth

Antifreeze: ethylene glycol competes for active site of alcohol dehydrogenase

• Ethylene glycol is metabolized to oxalic acid, which crystallizes in kidneys and causes renal
failure
• Can be treated by alcohol infusion
Simplified cases: Noncompetitive inhibition
• Since noncompetitive inhibitors do not
interfere in the binding of the
substrate (the dissociation constant of
ES and ESI have the same value Ks)
 Km is not affected

• However, increasing [S] can not abolish the inhibition  (ESI)

complex are formed and these are incapable of progressing to
reaction products
• The effect of a noncompetitive inhibitor is to reduce [ES] that can
advance to product
• Since Vmax = k2[Et], and the concentration of competent Et is
diminished by the amount of ESI formed
 Vmax is decreased
Simplified cases: Noncompetitive
inhibition
Examples of noncompetitive inhibitors

• Heavy metals like lead, mercury (breaks disulfide bonds), chromium will act
as non-competitive inhibitors

• Mono-amine oxidase (MAO) inhibitors that are used as anti-depressants:

They covalently react with the enzyme in the liver and effectively remove it.

• There are many potent drug interactions with MAO inhibitors. One of these is
tyramine, a compound that is present in red wine and aged cheeses
• MAO inhibitors and tyramine (blocks neurotransmitter reuptake in the brain)
 hypertensive crisis
• Patients are still subject to hypertension for as long as two weeks after
discontinuing the drug
Simplified cases: Uncompetitive inhibition

• The ES complex dissociates the

substrate with a dissociation constant
equal to Ks, whereas the ESI complex
does not dissociate it (i.e has a Ks
value equal to zero)
 Km is decreased

• Increasing [S] leads to increasing [ESI] (a complex incapable of

progressing to reaction products), therefore the inhibition can not
be removed
 Vmax is decreased
Simplified cases: Uncompetitive inhibition
Examples of uncompetitive inhibitors
• Lithium and the phosphoinositide cycle: an example of
uncompetitive inhibition and its pharmacological
consequences
Nahorski SR, et al Trends Pharmacol Sci. 1991 Aug;12(8):297-303
• The ability of lithium to exert profound and selective
psychopharmacological effects to ameliorate manic-depressive
psychosis has been the focus of considerable research effort.
• There is increasing evidence that lithium exerts its therapeutic action
by interfering with polyphosphoinositide metabolism in brain and
prevention of inositol recycling by an uncompetitive inhibition of
inositol monophosphatase
Examples of uncompetitive inhibitors
Kamali and Rawlins, Biopharmaceutics & Drug Disposition, 13(6), 403-409

• The effects of probenecid on zidovudine (a potent HIV

inhibitor) glucuronidation were investigated, in vitro, using
human liver microsomal preparations
• The presence of probenecid:
• reduced the Vmax for zidovudine glucuronide formation by more than
60 %
• reduced Km by 47 %
• uncompetitive inhibition
Hallmarks of reversible inhibitors
• When inhibitor concentration ↓es, enzyme activity is regenerated

• They bind to enzymes by non-covalent forces

• Inhibitor maintains a reversible equilibrium with the enzyme

Ki = equilibrium constant for dissociation of
Enzyme Inhibitor complexes
Lineweaver-Burk (L-B) plots of inhibited enzymes
Classes of reversible enzyme inhibitors…..
REGULATION OF ENZYME
SYSTEMS IN LIVING
SYSTEMS
 Regulation of Enzyme activity in living
systems
• Allosteric regulation
• Activation of latent enzyme
• Compartmentation of metabolic pathways
• Control of enzyme systems
• Enzyme degradation
• Isozymes
 Allosteric regulation
• Regulation of enzymes by small molecules that bind to a site distinct
from the active site;

• Binding of these small molecules changes the conformation and

catalytic activity of the enzyme

• Small molecules are called allosteric effectors

• Enzymes that are regulated by this mechanism are called allosteric
enzymes
 Mechanism of allosteric regulation
• Effectors alter enzymatic activity in 2 ways:

- ↑ ing or ↓ing Km OR

- ↑ ing or ↓ing Vm
 Allosteric effectors vs Allosteric enzymes

Allosteric effectors Allosteric enzymes

• K- type effectors • K- class allosteric enzymes
- Double reciprocal plots similar to
• V-type effectors competitive inhibition
- .e.g. phosphofructokinase
• V- class allosteric enzymes
-double reciprocal plots similar to
non-competitive inhibition
-e.g Acetyl CoA carboxylase
 Heterotopic effectors vs homotropic effectors

• Heterotopic effectors • Homotropic effectors

-bind at allosteric sites as
activating or inhibiting effectors
- e.g cAMP
- They are not identical to the
substrate
- substrates acting as effectors
- Bind to catalytic site
- Transmits an activity-modulating
effect to other subunits of the
enzyme molecule
 Allosteric enzymes
• Most are oligomeric (consisting of multiple subunits)
• Subunits may be identical or different and account for unusual kinetic
properties
• Generally located at or near branch points in metabolic pathways
• Direct substrates along one or more available metabolic paths
• Give a sigmoidal curve when the velocity (v) is plotted against the
substrate concentration (S)
• Exhibit cooperativity (↑ed enzyme affinity for substrate as a function
of substrate loading
Conformational changes in allosteric enzymes

• Non-covalent reversible binding of effector molecule at allosteric site

brings about a conformational change in the active site of the
enzyme;

• This leads to activation or inhibition of the enzyme

Allosteric enzymes
• Allosteric enzymes occur in 2
states
- T (tense or taut ) AND
R (relaxed) states;
- T and R states are in equilibrium
• Allosteric inhibitors favor T state
• Allosteric activators favor R state
Effect of substrate concentration on
allosteric and normal enzyme
Feedback inhibition
• End product of a pathway
accumulates, binds to and
inhibits a critical enzyme
upstream;
• either as a competitive inhibitor
or an allosteric effector
• Important in cell economy as it
averts synthesis of an already
available compound
 Activation of latent enzymes
• Some enzymes are synthesized as proenzymes (zymogens) and
undergo irreversible covalent activation by one or more peptide
bonds;
-E.g Chymotrypsinogen, Tripsinogen, Pepsinogen, Plasminogen etc
-Activation is brought about by specific ions or by other enzymes

-Zymogens provide a protective mechanism to prevent autodigestion of

tissue producing the digestive enzymes and or to prevent blood
coagulation
 Activation of latent enzymes…………
• Specific cleavage of zymogens causes conformational changes that
expose the enzyme active site

• Proteolytic enzymes(e.g. trypsin and pepsin) are inactivated by

inhibitor proteins that tightly bind to the enzyme active site

• Pancreatic trypsin inhibitor binds to and inhibits trypsin

• α- antiproteinase primarily inhibits elastase
 Activation of latent enzymes
• Another group of enzymes occur in interconvertible active and
inactive forms depending on cell needs
• Interconversion is via reversible covalent modification usually by
protein phosphorylation or dephosphorylation;
- This mechanism is rapid, reversible, and economical
- Saves energy as it does not require new protein synthesis or altered
gene expression
- Reversal of phosphorylation state restores the original condition
without the cost of degrading or replacing the protein
Activation of latent enzymes………..
Glycogen phosphorylase activity
regulation by covalent modification
Glycogen phosphorylase is a
homodimer
• Interconversion is via
phosphorylation and
dephosphorylation
• Some enzymes are active in the
dephosphorylated state and
become inactive in the
phosphorylated state
• E.g Glycogen synthase and Acetyl
CoA carboxylase
 Control of enzyme synthesis
• ↑Enzyme synthesis→ ↑ amount of enzymes → directly affects the rate
of reaction catalyzed by the enzyme
• Enzyme synthesis is regulated by genes via Enzyme induction and
Enzyme repression;
• These processes determine the concentrations of the enzymes at the
gene level through the mediation of hormones or other substances
• Levels of constitutive enzymes (house-keeping enzymes )are not
controlled and remain fairly constant
• Levels of Adaptive enzymes ↑ or ↓ as per body needs and are well
regulated
 Enzyme induction and repression
Enzyme induction
• ↑ed synthesis of enzyme
• E.g Insulin induces the synthesis
of Glucokinase, PFK,and
Pyruvate kinase
• Cortisol induces Pyruvate
carboxylase
Enzyme repression
• ↓ed synthesis of enzymes
• E.g. substrate can repress the
synthesis of enzyme;
• Glucose can repress Pyruvate
carboxylase
 Enzyme degradation
• Enzymes don’t last forever;

• They have varying half lives

• Many rate limiting enzymes have short half lives

• This is important in the efficient regulation of enzyme levels

 Isozymes
• Multiple forms of one enzyme that catalyze the same reaction but
differ in amino acid composition
• May be isolated from the same or different tissues
• Because of their structural differences they may be distinguished by
separation in an electric field (electrophoresis) or by reactivity with
selective antibodies
• They differ in Km, Vm or both for the same substrate
• E.g. CK, LDH
Isozymes……….
• Hexokinase • Glucokinase
• Has a low Km (high affinity) for • High Km(low affinity) for glucose
glucose; about 5 x 10-5 M ;about 2 x 10-2 M
• Fully saturated at blood glucose • Operates within the liver
concentration of 5 × 10-3 (mM).
• Activity changes in response to
• Activity changes little with changes in glucose
changes in glucose concentration
concentration
• Located in the muscle
Diagnostic enzymes and clinical relevance

• Quick and accurate diagnosis is a major characteristic of clinical

management
Enzymes in the pathological diagnosis
• Serum levels of many enzymes have diagnostic significance

• Enzymes in serum indicates that tissue or cellular damage has

occurred and components of the cell are released into the blood.

• Estimation of levels of an enzyme in a tissue e.g blood is referred to as

ENZYME ASSAY
• One commonly assayed group of enzymes are aminotransferases
Aminotransferases (AST,ALT)
• Alanine transaminase, ALT (serum glutamate-pyruvate transaminase)
and Aspartate transaminase, AST (serum glutamate-oxaloacetate
transaminase, SGOT) are commonly assayed
• Humans have cytosolic AST and a mitochondrial AST that function as
homodimeric enzymes
• Have application in the diagnosis of liver disease
Diagnosis of liver disease
• Typical liver enzymes measured are aspartate transaminase (AST) and
alanine transaminase (ALT).
• ALT is particularly diagnostic of liver involvement as this enzyme is found
predominantly in hepatocytes.
• ALT and AST ratio of the level of these two enzymes have diagnostic
value .
• Normally in liver disease or damage that is not of viral origin the ratio of
ALT/AST is less than 1.
• In viral hepatitis the ALT/AST ratio will be greater than 1.
• Measurement of AST is useful not only for liver involvement but also for
heart disease or damage
Diagnosis of liver disease………..

• Gamma-glutamyltranferase (GGT, γ-glutamyltransferase; also

called gamma-glutamyl transpeptidase) is involved in
glutathione (GSH) metabolism and also in amino acid transport
across membranes.
• Assessment of GGT levels in the blood is diagnostic for
diseases of the liver and the biliary system as well as disease of
the pancreas.
Diagnosis of myocardial infarction
• cardiac troponin I (cTnI),
• lactate dehydrogenase (LDH)
• creatine kinase (CK, also called creatine
phosphokinase, CPK)
• are commonly measured in the diagnosis of
cardiac infarct
Diagnosis of myocardial infarction……
• The level of AST elevation in the serum is directly proportional to the
number of cells involved as well as on the time following injury that the
AST assay was performed.
• Following injury, levels of AST rise within 8 hours and peak 24–36 hours
later.
• The level of AST should return to pre-injury levels within 3–7 days ,
provided a continuous insult is not present or further injury occurs.
• Measurement of AST is not, in and of itself, diagnostic for myocardial
infarction, taken together with LDH and CK measurements (see below)
• The level of AST is useful for timing of the infarct.
Diagnosis of myocardial infarction…
• Cardiac markers; cardiac troponins
• Troponins are complexes composed of three regulatory proteins, troponin C (TnC), troponin I
(TnI), and troponin T (TnT) attached to tropomyosin
• They are found in the grooves between thin actin filaments in striated muscle tissue
• The troponins are found in skeletal and cardiac muscle but not smooth muscle.
• Cardiac troponins found in the serum, specifically troponin I and T, are excellent markers for
myocardial infarction as well as for any other type of heart muscle damage.
• The measurement of plasma troponin I levels are highly diagnostic of necrosis of cardiac
muscle.
• Serum levels of troponin I rise within 4-8 hrs after the onset of chest pains caused by
myocardial infarction.
• The levels peak within 12-16 hrs after the onset of infarction and return to baseline within 5-9
days.
Diagnosis of myocardial infarction…
• Lactate dehydrogenase

• The measurement of lactate dehydrogenase (LDH) is especially diagnostic

for myocardial infarction because this enzyme exists in five closely related,
but slightly different forms (isozymes).

• Five isoforms of this enzyme are generated by combinations of two

different subunits encoded by two different genes. The subunits are
identified as the M form for muscle-specific (encoded by the LDHA gene)
and the H form for heart-specific (encoded by the LDHB gene).
Diagnosis of myocardial infarction…
• Lactate dehydrogenase
•The five types of LDH used in diagnosis and their normal distribution and levels in non-
disease/injury are listed below.

• LDH 1 (H4) – Found in heart and red-blood cells and is 17% – 27% of the normal serum total.

• LDH 2 (H3M1) – Found in heart and red-blood cells and is 27% – 37% of the normal serum total.

• LDH 3 (H2M2) – Found in a variety of organs and is 18% – 25% of the normal serum total.

• LDH 4 (H1M3) – Found in a variety of organs and is 3% – 8% of the normal serum total.

• LDH 5 (M4) – Found in liver and skeletal muscle and is 0% – 5% of the normal serum total.
Diagnosis of myocardial infarction
• Following a myocardial infarct the serum levels of LDH rise within 24-
48 hours reaching a peak by 2–3 days and return to normal in 5-10
days.
• Especially diagnostic is a comparison of the LDH-1/LDH-2 ratio.
Normally, this ratio is less than 1. A reversal of this ration is referred to
as a "flipped LDH".
• Following an acute myocardial infarct the flipped LDH ratio will appear
in 12–24 hours and is definitely present by 48 hours in over 80% of
patients.
• Also important is the fact that persons suffering chest pain due to
angina only will not likely have altered LDH levels.
Creatine kinase
• Creatine kinase (CK, or creatine phosphokinase, CPK) is found primarily in
heart and skeletal muscle as well as the brain.
• Therefore, measurement of serum CPK levels is a good diagnostic for
injury to these tissues.
• The levels of CPK will rise within 6 hours of injury and peak by around 18
hours. If the injury is not persistent the level of CK returns to normal within
2–3 days.
• Like LDH, there are tissue-specific isoforms of CPK derived from the
expression of two distinct creatine kinase genes. The muscle CPK isoform
is expressed from the creatine kinase, muscle (CKM) gene while the brain
isoform is expressed from the CKB gene.
Creatine kinase
• Dependent upon the tissue of expression, three major CPK isoforms are found in human
tissues.
• CPK3 (CPK-MM) is a homodimer of two CKM encoded proteins and is the
predominant isoform in muscle and is 100% of the normal serum total.

• CPK2 (CPK-MB) is a heterodimer of the CKM and CKB encoded proteins and this
form accounts for about 35% of the CPK activity in cardiac muscle, but less than 5% in
skeletal muscle and is 0% of the normal serum total.

• CPK1 (CPK-BB) is a homodimer of two CKB encoded proteins and is the

characteristic isoform in brain and is in significant amounts in smooth muscle and
several other tissues and is 0% of the normal serum total.
Carbonic anhydrases
• catalyze the formation of carbonic acid (H2CO3)
from CO2 and H2O,

• are useful both as pharmacologic targets and as

diagnostic tools in certain disease states.
 Importance of laboratory tests
• Disease diagnosis
• To monitor disease progression
• To assess risk
• To inform prognosis
• For population screening prognosis
 Enzymes in circulation

• Plasma specific or plasma • Non- plasma specific or plasma

functional enzymes non functional enzymes
Plasma specific or plasma functional enzymes
Non-plasma specific or plasma non- functional
enzymes
Non-plasma specific or plasma non- functional
enzymes
Causes of raised levels of enzymes
• Cellular damage
• Increased cell turnover
• Proliferation of cells
• Increased synthesis of enzymes
Causes of decreased plasma enzymes activities

• Decreased enzyme synthesis

• Congenital deficiency
Decrease in plasma enzymes seen in certain diseases

• Amylase
• Liver disease
• Pseudocholinesterase
• Viral hepatitis,malnutrition,liver
• Glucose 6- phosphate cancer,cirrhosis of liver
dehydrogenase (G6PD) in rbc

• Congenital deficiency with

hemolytic anemia
Diagnostic enzymes
Diagnostic enzymes
Diagnostic enzymes (contd)
Diagnostic enzymes…………
Diagnostic enzymes (contd)
Diagnostic enzymes………….
Diagnostic enzymes……..
A note on PSA
• PSA- Protein specific antigen

• Not an enzyme

• Normal levels (1-4ng)

• Reliable marker in detection of prostate cancer

 THIS MATERIAL IS FROM AN UNAUTHORIZED SOURCE.
I DO NOT OWN THE RIGHT TO THE INFORMATION ON THIS
SLIDES