Isoenzymes

Isoenzymes
• Multiple forms of enzymes (isoenzymes) occur
which have similar catalytic activities but
different structures.

• Different isoenzymes are often organ-specific

and their determination may improve the
specificity of enzyme tests.

• The heterogeneity of some isoenzymes is due to

different protein subunits which are coded for by
separate genes.
Isoenzyme: Differences
• Charge: Can be separated by electrophoresis
• Stability to heat denaturation
• Reaction to chemical inhibitors
• Affinity for substrates or coenzymes
• The most common ones: LDH, CK, ALK and ACP
Reasons for isoenzyme
• Synthesized from different genes (malate
dehydrogenase in cytosol versus in mitochondria)

• Oligomeric forms of more than one type of

subunits (lactate dehydrogenase)

• Different carbohydrate content (alkaline

phosphatase)
Lactate dehydrogenase (LDH)

• 5 isoenzymes, LDH1 – LDH5

• Tetramer
▫ M subunits (M for muscle), basic
▫ H subunits (H for heart), acidic
• Different catalytic activities
• Used as the marker for disease diagnosis
Lactate dehydrogenase (LDH)
isoenzymes with slightly different subunits
Isoenzyme Composition Composition Present in Elevated in
name
LDH1 ( H4) HHHH Myocardium myocardial
, RBC infarction

LDH2 (H3M1) HHHM Myocardium

, RBC
LDH3 (H2M2) HHMM Kidney,
Skeletal
muscle
LDH4 (H1M3) HMMM Kidney,
Skeletal
muscle
LDH5 (M4) MMMM Skeletal Skeletal
muscle, muscle and
Liver liver diseases
Creatine phosphokinase
(CK, CPK)
Creatine phosphokinase (CK, CPK)
• CK occurs in 3 isoenzymes, each is a dimer
composed of 2 subunits [M (muscle) or B
(brain)]: CK1 = BB from brain, CK2 = MB from
the heartand CK3 = MM from skeletal muscle.

• Each CK isozyme shows a characteristic

electrophoretic mobility.
Creatine phosphokinase (CK, CPK)
• CPK2 is undetectable (<2%) in serum for healthy
individuals, and elevated to 20% in the first 6-18
hrs after myocardial infarction.

• Used as a earliest reliable indicator of

myocardial infarction.
Creatine phosphokinase (CK, CPK)
Isoenzyme
Composition Present in Elevated in
name

CK-1 BB Brain CNS diseases

Acute
Myocardium/
CK-2 MB myocardial
Heart
infarction
Skeletal
CK-3 MM muscle,
Myocardium
Alkaline phosphatase (ALP)

• Maximal activity at a high pH 9.0-10.5

• Widely distributed throughout the body

• High levels are seen is liver, bone, placenta

and intestine and useful to assess
hepatobiliary and bone diseases
ALP Isoenzymes
• Humans and most other mammals contain the
following ALP isoenzymes:
▫ ALPI – intestinal
▫ ALPL – tissue non-specific (liver/bone/kidney)
▫ ALPP– placental (Regan isozyme)
• All mammalian ALP isoenzymes except placental
are inhibited by homoarginine.
• Heating for ~2 hours at 65°C inactivates most
isoenzymes except placental isoforms.
Pyruvate Dehydrogenase
Complex (PDC)
(Multienzyme Complex)
Pyruvate Dehydrogenase Complex
(PDC)

• large complex containing many

copies of each of 3 enzymes, E1,
E2, & E3.

▫ The inner core of mammalian PDC

is consisting of 60 copies of E2.
▫ At the periphery of the complex are:

30 copies of E1 (itself a tetramer with subunits a2b2).

12 copies of E3 (a homodimer), plus 12 copies of an E3
binding protein that links E3 to E2.
Pyruvate Dehydrogenase Subunits

Enzyme Abbreviated Prosthetic Group

Pyruvate E1 Thiamine
Dehydrogenase pyrophosphate (TPP)

Dihydrolipoyl E2 Lipoamide
Transacetylase

Dihydrolipoyl E3 FAD
Dehydrogenase
DNA polymerase (Multifunctional
Enzyme
• Catalyzes the polymerization
of deoxyribonucleotides into
a DNA strand.
• Uses a magnesium ion for
catalytic activity.
• DNA polymerases have
highly-conserved structure,
which means that their
overall catalytic subunits
vary, on a whole, very little
from species to species.
Function
• DNA polymerase can add free nucleotides to
only the 3’ end of the newly-forming strand. This
results in elongation of the new strand in a 5'-3'
direction.

• Error correction(proof reading and repair) is a

property of some, but not all, DNA polymerases.
This process corrects mistakes in newly-
synthesized
Different DNA polymerases have
different functions in the cell
Eukaryotic DNA Polymerases