ENZYMES IN MEDICINEENZYMES IN MEDICINE

• Diagnostic indicators – the activities of many enzymes are routinely determined in plasma ( rarely in tissue biopsies) for diagnostic purposes in diseases of the heart, liver, skeletal muscle, pancreas and other tissues - enzyme diagnostics

• Therapeutic agents – several enzymes are used as drugs; new approach - enzymotherapy

• Diagnostic tools – use as chemicals in clinical laboratory assays

ENZYMES IN CLINICAL DIAGNOSIS

secretory - produced by tissues (namely liver), acting in plasma – prothrombin, plasminogen, cerruloplasmin, choline esterase; lipoprotein lipase

Enzymes

intracellular – function intracellulary, have no physiological use in plasma

- membrane bound – ALP, GMT

- cytosolic – ALT, AST, LD, MDH

- mitochondrial – AST, GMDH - lysosomal - ACP

- tissue specific – glucose-6-phosphatase – liver amylase – pancrease

LD1 – heart

• Healthy individuals - levels of intracellular enzymes fairly constant, low –

the rate of enzyme release from damaged cells into plasma balanced by the rate of removal of enzyme protein from plasma

Physiological enzyme levels reference values of the enzyme activities

(determined in clinical laboratory – each lab has its own reference values)

• Elevated enzyme activity in the plasma – reflect tissue damage accompanied by increased release of intracellular enzyme

Skeletal muscle during exertion – physiologically elevated levels of muscle enzymes in plasma

• Many diagnostically important enzymes = isoenzymes – pattern of isoenzymes in plasma (determined electroforetically)

– a means of identifying the damaged tissue

ALTERATION OF ENZYME PLASMA LEVELS

Increased values – increased cell membrane permeability

anoxia, disturbances of energy metabolism cytosolic enzymes – ALT, LD, CK

- cell necrosis membrane-bound enzymes – ALP, GMT mitochondrial enzymes – AST, GMDH

- induction of the enzyme synthesis drugs – ALP, GMT

Decreased values – inhibition of the activity drugs

- inhibition of the synthesis cell damage, drugs

Examples of enzymes commonly assayed for diagnostic purposes

Enzyme Location Cause of elevated plasma level

Acid phosphatase - ACP Prostate Prostatic cancer

Alkaline phosphatase – ALP Bone, liver Rickets, hypoparathyroidism, osteomalacia, obstructive jaundice, cancer of bone/liver

Alanine aminotransferase – ALT Liver (muscle, Hepatitis, jaundice, circulatory heart, kidney) faillure with liver congestion

Aspartate aminotransferase – AST Heart, muscle, Myocardial infarction, muscle red cells, liver damage, anemia, hepatitis,

circulatory faillure with liver congestion

Amylase - AM Pancres Acute pancreatitis, peptic ulcer

-Glutamyl transferase – GMT Liver, kidney, Hepatitis, alcoholic liver pancreas damage, cholestasis

Examples of isoenzymes commonly assayed for diagnostic purposes

Enzyme Location Cause of elevated plasma level

Creatine kinase – CKCK-MB Heart Myocardial infarctionCK-MM Skeletal muscle Muscular dystrophy

Lactate dehydrogenase – LDLD1 > LD2 Heart, kidney, Myocardial infarction, kidney blood cells disease, megaloblastic anemia,

leukemia

LD2, LD3 LeukemiaLD5 Liver, muscle Liver disease, muscle damage

ENZYMES IN THERAPY

• Substitution of missing production of digestive enzymes – digestive enzymes – pepsin trypsin…

• Removal of deposits of death tissue or fibrin (e.g. in lungs, eyes), treatment of skin defects – proteinases, nucleases, collagenase

• Acceleration of fibrinolysis in lungs embolization (activation of plasmin

and plasminogen) – streptokinase, urokinase

ENZYMOTHERAPYOrally administered enzymes – treatment of a variety disorders - digestive, gastrointestinal, pancreatic - inflammatory diseases, edema - immune and autoimmune diseases

(arthritis, multiple sclerosis) - viral diseases (herpes, AIDS) - cancer

Mixtures of enzymes of plant and/or animal origin - proteinases, amylase, lipase - administered as acidoresistent tablets

• Pancreatin – trypsin, chymotrypsin, lipase, amylase• Wobenzym – pancreatic and plant proteolytic enzymes – trypsin,

chymotrypsin, papain (Carica papaya), bromelain (ananas) = combination of enzymes with different specificity, pH optimum, stability, interaction with inhibitors and antiproteinases

multiple action

• Mechanism of resorption (transport of large macromolecules across the intestinal barrier) – paracellular transport, receptor mediated endocytosis and transcytosis

• Mechanism of action – interaction with plasma antiproteinases –

1-antitrypsin, 2-macroglobulin complexes

direct proteolytic action, degradation of adhesive

molecules, secretion of cytokins (tranforming growth factor TGF-), modulation of receptor function

not fully clarified

?

ENZYMES - USE IN LABORATORY ASSAYS

Enzymes isolated from different sources - used for determination of various substances in the blood, plasma/serum and urine enzyme methods

much more specific than chemical methods, the presence of relative substances with similar chemical properties does not hinder

Components of commercial kits or diagnostic strips

- determination of glucose - glucose oxidase, peroxidase

cholesterol - cholesterol esterase, cholesterol oxidase

peroxidase,

urea – urease, ……. in blood, plasma, serum

- proof of glucose (glucose oxidase), …….. in blood or urine (strips)

Markes in the immunochemical analysis

- ELISA (=enzyme-linked immunoadsorbent assay) – peroxidase, alkaline phosphatase

NUCLEOTIDESNUCLEOTIDESStructure, FunctionStructure, Function

NUCLEOTIDE STRUCTURENUCLEOTIDE STRUCTURE

Nucleotides

nitrogenous base + pentose + phosphate group(s)

purine ribose 1 - 3

pyrimidine deoxyribose

other (nicotinamide)

Nucleosides

NC

C

CCN

N

NH

CH

NH2

|

H

NC

C

CCN

N

NH

CH

O||

H

H2N

NC

C

CCNH

O

H

H

O|| CH3

adenineadenine guanineguanine

cytosinecytosine thyminethymine uraciluracil

THE NITROGENOUS BASESTHE NITROGENOUS BASESTHE NITROGENOUS BASESTHE NITROGENOUS BASES

NC

C

CCNH

O

NH2

|

H

H NC

C

CCNH

O

O||

HH

H

Purine bases

Pyrimidine bases

NUCLEOSIDENUCLEOSIDENUCLEOSIDENUCLEOSIDE

• A sugar - base combination.

OHOCH2

H HHH

OH H

BaseBase

SugarIn this case deoxyribose

SugarIn this case deoxyribose

-N-glycosidiclinkage

-N-glycosidiclinkage

N

O

H HHH

OH H

HOCH2 O

H HHH

OH OH

HOCH2

O

H HHH

OH

HOCH2

NC

C

CCNO

NH2

|

H

H

NC

C

CCNO

H

H

CH3

O

NC

C

CCNO

H

H

O

H

OH

thymine uracil

cytosine

deoxythymidine uridine

cytidine

O

H HHH

OH H

O

H HHH

OH

O

H HHH

OH

HOCH2

HOCH2

HOCH2

OH

OH

NC

C

CCN

N

N

CH

NH2

|

H

NC

C

CCN

N

N

CH

OH|

H

NC

C

CCN

N

N

CH

O

H

H2N

adenine guanine

hypoxanthine

deoxyadenosine guanosine

inosine

!

NUCLEOTIDESNUCLEOTIDESNUCLEOTIDESNUCLEOTIDES

5’-OH on the sugar of a nucleoside is converted into a phosphate ester.

NC

C

CCN

N

N

CH

NH2

|

H

O-O-P-O-CH2

H HHH

OH H

|

O- -

O||

deoxyadenosine monophosphate(dAMP)

deoxyadenosine monophosphate(dAMP)

Each is named based on sugar and base nameand then the number ofphosphates is indicated.

adenine

phosphate chain

ribose

CH2 O

OH OH

N

NN

N

NH2

OPOPOPO-

O

O-

O

O-

O

O-

ATPATP - - adenosine triphosphateadenosine triphosphate

ATPADP

AMP

NUCLEOTIDE FUNCTIONNUCLEOTIDE FUNCTION

• Precursors of DNA, RNA - NTPs

• Energy transport - ATP

• Allosteric effectors of enzymes – ATP, ADP, AMP

• Covalent modification of enzymes – ATP

• Intracellular mediators (= second messengers) – cAMP, cGMP

• Coenzymes – NAD+, NADP+, FAD, CoA-SH

• Activated precursors of polysaccharaides, glycoproteins, proteoglycans, phospholipids, glycolipids – UDPG, UDPGA, UDPGal…, CDP-choline, CDP-diacylglycerol…

• Active groups (group transport) – SAM, PAPS

NAD+NAD+

OCH2

OH OH

OPO

O-

O

OCH2

OH OH

OPO

O-N

NN

N

NH2

N+

C

O

NH2

reactivesite

nicotinamide

adenine

ribose

OCH2

OH OH

OPO

O-N

NN

N

NH2

O

C HH

CH OH

CH OH

CH OH

CH H

N

NH3C

H3C N

NH

O

O

ribose

adenine

riboflavin

reactive site

FAD FAD

Coenzyme ACoenzyme A

phosphorylated ADP

pantothenateunit

CH2 O

O OH

N

NN

N

NH2

OPOPO

O-

O

O-

P O-O

O-

C-CH2-CH2-N-C-C-C-CH2

OO H

CH3HO

CH3

HH-N

CH2-CH2

SSulfhydryl

group

O

H

H

OH

O H

OHOH

H

OHH

OH

CH2OH

H -D-glucose -D-glucose

OH

OH

H

H

H

OH

CH 2 OH

H

OH

OH OH

H

H

H

OH

CH 2 OH

H

OH

O

Non-polar tail

O CO

O CO

C

C

CH2

H

H

H

PO

O

O-

2-O-

cAMP

cAMP – cyclic adenosine monophosphate

NC

C

CCN

N

N

CH

NH2

|

H

O

H HHH

H

-

P O

O

O

O-

O

CH2

cAMP – cyclic adenosine monophosphate

-intracellular mediator, second messenger of hormonal signal tranduction via adenylate cyclase cascade- mechanism of action: allosteric effector

CH2-O-C

HO HOHO

C-O-CH

CH2-O - P

O

OO

SCoA

HO

O

SCoA

HOHO

P~