KONGUNADU ARTS AND SCIENCE COLLEGE (AUTONOMOUS)

Coimbatore – 641 029 DEPARTMENT OF BIOCHEMISTRY (PG)

C.P.3 Enzymes and Enzyme Technology

Dr.Nithya devi

Associate professor

Department of Biochemistry (PG)

Isoenzymes

ISOENZYMES

2

Isoenzymes or isozymes are mutipleforms of same enzyme that catalysethe same chemical reaction

Different chemical and physical properties:

Electrophoretic mobility

Kinetic properties

Amino acid sequence

Amino acid composition

S.

No

Property E.g.

1 Electrophoretic

mobility

Isoenzymes of Lactate dehydrogenase have

different electrophoretic mobility

2 Heat stability Alkaline phosphatase isoenzymes are either

heat labile or stable

3 Inhibitor An inhibitor can inhibit only one isoenzyme

of an enzyme eg. Acid phosphatase

4 Km Glucokinase and hexokinase

5 Cofactors Mitochondrial isocitrate dehydrogenase

requires NAD+ , cytosolic form requires

NADP+

6 Tissue localisation LDH 1 is present in heart, LDH 5 in muscle

7 Antibodies For creatine kinase, each isoenzyme can be

bound only by a specific antibody

3

Lactate dehydrogenase (LDH)

4

E.C – 1.1.1.27 L-lactate :NAD+ oxidoreductase:LDH

Molecular weight- 134 kDa

tetramer

M (A) -muscle –chromosome 11(basic)

H (B) -heart – chromosome 12(acidic)

Lactate dehydrogenase (LDH)

Normal values

Serum -100 -200 U/L

CSF - 7 -30 U/L

Urine - 40 -100 U/L5

Isoenzyme

name

Composition Electrophore

tic migration

Present in Elevated in

LDH 1

Heat

resistant

( H4) Fastest

moving

Myocardium,

RBC,kidney

myocardial

infarction

LDH2

Heat

resistant

(H3M1) Myocardium,

RBC,kidney

Kidney

disease,megalo

blastic anemia

LDH3 (H2M2) brain Leukemia,malig

nancy

LDH4

Heat labile

(H1M3) Lung,spleen Pulmonary

infarction

LDH5

Heat labile

Inhibited by

urea

(M4) Slowest

moving

Skeletal

muscle, Liver

Skeletal muscle

and liver

diseases

6

Lactate dehydrogenase (LDH)This is an example in which two duplicated genes have

become specialized to different tissues.

The isozymes are also differentially expressed in

different developmental stages. Before birth the heart

is more anaerobic compared with adulthood. Indeed,

before birth the main isozyme in the heart is the M4,

and with time it switches to HM3 (at birth), to H2M2

and HM3 at 1 year after birth, and to H3M AND H4

after 2 years.My main LDH is HM3. Great!

My main LDH is HM3

7

Atypical forms of LDH

8

sixth isoenzyme LDH- X

Seventh isoenzyme – LDH -6

Clinical significance of LDH

9

Myocardial infarction (LDH 1>LDH2)

Megaloblastic anemia (50 times upper limit of LDH 1 and LDH 2)

Muscular dystrophy (LDH 5)

Toxic hepatitis with jaundice (10 times more LDH 5)

Renal disease- tubular necrosis or pyelonepheritis

Pulmonary embolism LDH 3 (massive destruction of platelets)

Leukemia (LDH 2 and LDH 3)

Malignancy (LDH 3)

Hodgkins disease

germ cell tumors

Urinary LDH-3 to 6 times normal:

chronic glomerulonephritis Systemic

lupus erythematosus Diabetic

nephrosclerosis

Bladder and kidney malignancies

10

In CSF:

Bacterial meningitis – LDH 4 and LDH 5

11

Viral meningitis

Metastatic tumors

- LDH 1

- LDH 5

Neonatal cases of intracranial

haemorrhage associated with seizures

and hydrocephalus

LDH in starch gelThe H(B) monomer

is very negatively charged

12

CREATINE PHOSPHOKINASE

Adenosine triphosphate:creatine N-

phosphotransferase

E.C-2.7.3.2

Dimeric enzyme (82 kDa)

4 -60 IU/L

13

14

Enzyme unstable in serum

Activity lost due to sulfhydryl group

oxidation at active site

Dimer (each of 41000 Da)

B (brain) – chromosome 14 M (muscle) –chromosome 19

15

Isoenzy

me

name

Compo

sitionPresent in Elevated in

CK-1

Fast

moving

BB

Brain,prostate,GI

tract,lung,bladder,uteru

s,placenta

CNS diseases

CK-2

2% of

total

MB Myocardium/ HeartAcute myocardial

infarction

CK-3

Slow

moving

MMSkeletal muscle,

Myocardium

All 3 in cytosol 16

17

atypical forms of CK

18

Fourth form - CK-Mt (chromosome 15)

severe illness Malignant tumors

macroCK

type 1- CK BB complexed with

IgG

type 2-oligomeric CK-Mt

Clinical significance of CK

19

CK 1 elevated:

very low birth weight newborns brain

damage in neonates neurological

injury –CK 1 rise inCSF

>200 U/L –die100 – 200 U/L – survive with

neurological defecits

Elevated CK 1

20

Adenocarcinomas of GI tract

Carcinoma lung

Ca

prostate,bladder,testes,kidneys,breast,

ovaries,uterus,CNS,leukemia,lympho

ma and sarcoma

Elevated CK 2:

myocardial infarction head injuriessubarachnoid haemorrhage exerciseElevated CK 3:muscular dystrophies(DMD- 10000 IU/L)myopathies

hypothyroidism (5 fold more than normal value,also CK 2 is elevated)

21

Alkaline phosphatase (ALP)

22

E.C -3.1.3.1.

Orthophosphoric monoester

phosphohydrolase

In mucosa of small intestine, proximal

convoluted tubule, bone, liver,

placenta

Catalyses alkaline hydrolysis of

naturally occuring and synthetic

substrates

Isoenzymes of ALP

23

Alpha 1 ALP-epithelial cells of biliary canaliculi

Alpha 2 heat labile ALP- hepatic cells

Alpha 2 heat stable ALP-not destroyed at65˚C inhibited by phenylalanine

placental

Pre beta ALP – bone,heat labile Gamma ALP – intestinal cells inhibited by phenylalanine

Leukocyte alkaline phosphatase –decreased in CML increase in lymphoma

ATYPICAL ISOENZYMES

Regan isoenzyme-heat stable,inhibited by L-phenylalanine

Nagao isoenzyme- variant of regan

inhibited by L-leucine

Clinical significance

24

Hepatobiliary disease

Hepatic carcinoma

Hepatic metastases

Pagets disease (10 – 25 times) Bone cancer

Healing of bone fracture

Osteomalacia and rickets

Hyperparathyroidism

Ca of ovary,uterus-regan isoenzyme

Metastatic Ca of pleural surfaces –Nagao isoenzyme

Acid phospatases

25

acid phosphatses E.C -3.1.3.2.Hydrolyse phosphoric acid ester at pH 5 -6 In lysosomesExtalysosomal-prostate,bone,spleen,platelet, liver,kidney (pH - 5)RBC (pH – 6)

0 – 0.6 U/LExtremly heat labile

Isoenzyme of ACP

prostatic

26

Erythrocytic

inhibitor

dextrorotatory

tartarate ions

formaldehyde

cupric ions

Majorly the serum contains tartarate

resistant ACP (originating in

osteoclasts)

Clinical significance

27

To detect, monitor Ca prostate

Tartarate resistant ACP increase in

pagets disease and bone cancer

Marker of bone disease-increases in:

giant cell tumor of bone

normal growing children Gauchers

disease

In high concentrations in semen

SERUM AMYLASE(calcium

metalloenzyme)

28

E.C -3.2.1.1.

Molecular weight -54 -62 kDa

From salivary gland and

pancreas

Enzymes are products of 2 closely

linked loci on chromosome 1

macroamylases

29

Serum aldolase

30

Tetramer

Catalyses interconversion of fructose-1,6-bi-phosphate and triose phosphate

5 isoenzymes

Subunits

A B

1- 7.5 U/L

Skeletal muscle,liver,brain,heart

Clinical significance of serum

aldolase

31

Elevated in:

Progressive muscular dystrophy

particularly high in DMD

Viral hepatitis

Advanced cancer of prostate

SOURCES

PLASMA

DERIVED/PLASM

A SPECIFIC

CELL

DERIVED/PLASM

A NON SPECIFIC

SECRETOR

YMETABOLIC

BLOOD COAGULATION

ENZYMES

FERROXIDASE

LIPOPROTEIN LIPASE

PSEUDOCHOLINESTERA

SE

32

Mechanisms responsible for

abnormal levels

Increased serum

level

decreased serum

level

Increased

release

Impaired

excretion

Decreased

formation

Enzyme

inhibition

genetic acquiredCell

necrosi

s

Increased

permeabilit

y33

34

Enzymes in blood

Cell

death

Defects in cellular membrane

Release of cytoplasmic

enzymes initially

In infarctions

35

Elevation of enzymes in blood

No.of cellinjured

Gradient ofcell/plasma

Rate ofenzymeentry inplasma

Rate of clearance

from plasma

36

Serum enzyme assay in clinical

practice

37

In diagnosis

In differential diagnosis

In prognosis

Early detection of disease

Serum transaminases

38

Serum transaminases

Catalyse interconversion of

aminoacids to ketoacids by transfer of

amino group

AST-aspartate

aminotransferases(SGOT)

10-30 U/L

ALT-alanine aminotransferases

(SGPT)

10-40 U/L

Both present in plasma,bile,CSF,saliva 39

Gamma glutamyl

transpeptidases (GGT)

40

E.C -2.3.2.2.

Γ – glutamyl – peptide:amino acid Γ –glutamyl transferases

In proximal convoluted

tubule,liver,pancreas,intestine

Clinical significance

hepatobiliary disease

neoplasms

heavy drinkers

cholinesterase

41

Hydrolyse acetylcholine

Types-

acetylcholinestarase -3.1.1.7

pseudocholinesterase – 3.1.1.8 Clinical significance

insecticide poisoning

atypical form of enzymes who are at risk to muscle relaxants

sensitive indicator of synthetic capacity of liver

Glucose – 6 –phosphate dehydrogenase

Dimer with identical subunits In HMP - for production of NADPH

G-6-P + NADP+ 6-PG + NADPH + H+

hemolytic anemia

prolonged neonatal jaundice

conditions are directly related to the inability of specific cell types to regenerate reduced nicotinamide adenine dinucleotide phosphate (NADPH)

42

Serum lipase

43

E.C – 3.1.1.3. Molecular weight -48 kDa

Hydrolyses glycerol esters of long

chain fatty acids

Pancreas, intestinal and gastric

mucosa

0.2 – 1.0 U/L

Serum enzymes in cardiac

diseasesWhy enzyme diagnosis?

Enzyme assays

Creatine phosphokinase (CPK)

Aspartate transaminases (SGOT OR AST)

Lactate dehydrogenase (LDH)

γ-Glutamyl transpeptidase (GGTP)

Histaminase

Pseudocholinesterase

44

Cardiac biomarkers in

myocardial infarction

45

Onset peak

durationCK-MB

Troponins

AST

Myoglobin

46

3-6 hrs 18-24 hrs 36-72

hrs

4-10hrs days 18-24 8-14

6-12hrs 24-48 hrs 6-8 days

24-36 hrs d 4-5days 10-12

1-4hrs 6-7hrs 24hrs

4.5-20% of total

LDHFlipped pattern

Serum enzymes in GI tract

diseasesSerum amylase

A cute pancreatitis-4 -6 fold increase in 2 -12 hrs ,maximum level 12 -72 hrs , normal in 3 - 4 day

Urinary amylase - increased on 1st day and remains elevated till 8- 10 day

Ca pancreas- amylase in ascitic and pleural fluid

Cholecystitis – 4 fold elevation

47

Serum lipase

Acute pancreatitis:

2 -50 times in 4 -8hrs,peaks at 24hrs,decreases in 8 -14 days

48

Serum enzymes in liver

disease

Serum transaminases

Severe toxic hepatitis

Extrahepatic cholestasis

Cirrhosis (AST>ALT)

49

Hepatic carcinoma (5 -10 fold rise)

Hepatobiliary disease

Extrahepatic obstruction (10- 15 times)

Serum alkaline phosphatases

Serum LDH

• Toxic hepatitis with jaundice (10 times more LDH 5)

5’nucleotidase

Gamma glutamyl transferase

Extrahepatic and intrahepatic causes (2 -6 fold incr

Early infectious hepatitis

50

Alcoholic cirrhosis and alcoholics

Hepatic carcinoma

Serum enzymes in muscle

diseases

CPK

SGOT/SGPT

51

Serum aldolaseProgressive muscular dystrophy

muscular dystrophies

myopathies

hypothyroidism (5 fold more than

normal value,also CK 2 is elevated)

Muscular dystrophy and dermatomyositis

Serum enzymes in bone

diseases

Alkaline phosphatase

• Pagets disease (10 – 25 times)• Bone cancer• Healing of bone fracture• Osteomalacia and rickets

Acidphosphatase

• Marker of bone disease-increases in:• giant cell tumor of bone• normal growing children

52

As tumor markers

53

Aldolase-liver

ALP – bone,liver,leukemia,sarcoma Placental ALP – ovarian,lung,hodgkins Amylase – pancreatic CPK BB –

prostate,lung,breast,colon,ovarian

GGT – liver LDH – liver,lymphoma,leukemia Neuron specific enolase – tumors of

neuroendocrine origin

Prostate specific antigen (PSA

or semenogelase)

54

From secretory epithelium of prostate gland

32 kDa glycoprotein

Mild Serine protease activity

1- 5 μg/LLevels between 4 -10 μg/L –increased risk

of prostate cancer

>10 μg/L - suggestive of Ca prostate>20 μg/L - Ca prostate with metastases

Enzymes-therapeutic agent

1.Streptokinase

plasminogen

streptokinase plasmin

fibrinsoluble product 2.urokinase

55

3. Bacterial asparginase in leukemia4. α- chymotrypsin-extraction of lens

5.Chymotrypsin,papain –antiinflammatory

6. Collagenase – debridment of dermal ulcers

7. Fibrinolysin – venous thrombosis, pulmonary embolism

8. Hyaluronidase – rapid absorption of drugs injected subcutaneously

56

9.Lysosome –antibacterial (eye infections)

10.Trypsin – clean wounds treatment of acute

thrombophlebitis

57

Analytical use of enzymes

as reagents

as labels

58

Alcohol dehydrogenase

Lactate dehydrogenase

Glucose oxidase and peroxidase

Uricase

Urease

Cholesteroloxidase andperoxidase

59

Ethanol

Lactate

Glucose

Uric acid

Urea

cholesterol

As labels

60

In immunoassays

for determining concentration of drugs,

hormones

Glucose -6- phosphate dehydrogenase

Alkaline phosphatase

Beta galactosidase

peroxidase