ENZYMES

Siv Milliscent E. Balbas, RMT

What are Enzymes?

CHON catalyst of chemical reactions and

catalyzes all metabolic reactions of cells (eg. Respiration, photosynthesis, digestion,)

biological catalyst specific for its substrate

Parts of an Enzyme
Active site- portion of the enzyme where

reaction takes place

Substrate- binds to Active site of an enzyme

Allosteric site
Other site besides the active site

SUBSRTATE

ENZYME

LOCK AND KEY FIT

SUBSRTATE

ENZYME

INDUCED FIT

 Cofactor
Nonprotein molecule which maybe necessary for

enzyme activity
Coenzyme- Organic cofactor (NAD, heme,

vitamins)
Activators- Inorganic cofactor (eg. Chloride or

magnesium ions, and other metals)

Cofactor
SUBSRTATE

ENZYME

APOENZYME

HOLOENZYME

 NOTE:
Holoenzyme- complete, active enzyme system Proenzyme (zymogen)- inactive enzyme

Isoenzyme- enzymes that have different physical properties but have the same catalytic functions

Classification of Enzymes
OXIDOREDUCTASES
Catalyze an oxidation-reduction reaction between

2 substrates

TRANSFERASES
Catalyze the transfer of a group from one

substrate to another

 HYDROLASES
Catalyzes hydrolysis of various bonds

LYASES
Removal of groups without hydrolysis

ISOMERASES
Catalyze the interconversion of geometric, optical

or positional isomers

LIGASES
Catalyze the joining of two substrate molecules,

Factors the Influence Enzymatic Reactions

SUBSTRATE CONCENTRATION

pH ( 7.0- 8.0)
TEMPERATURE ENZYME CONCENTRATION

COFACTORS
INHIBOTORS
Competitive inhibitors Noncompetitive inhibitor Uncompetitive inhibitor

 Competitive Inhibitors
Competes with substrate for binding to active site

Non-Competitive Inhibitors
Does not bind to the active site, but removes

cofactors of the enzyme

Uncompetitive Inhibitors
Inhibitor binds to E-S complex preventing

dissociation

Enzyme Tissue Specificity

High
ACP (RBC, Prostate)

Moderate
AST (Liver, heart, skeletal muscle)) CK (Heart, skeletal muscle, brain) ALP (liver, bone, kidney)

Low
LD (In ALL tissues)

ALT (Liver)

AMS (Pancreas, salivary glands)

LPS (Pancreas)

Hepatic Enzyme Profile

ALP

AST
LD (LDH)

GGT
ChE

Alkaline Phosphatase (ALP)

optimum pH : 10 highest concentration in liver, kidney,

placenta, intestine, WBC

 Increase levels in:

Bone formation

Pagets disease
Rickets Osteoblastic tumors Cancer Sever fracture Liver disease

 Collection:
AVOID using citrate, oxalate, EDTA

Substrate: Organic phosphates (eg. glyceroPO4 and -nitrophenylPO4) Methods:

Bodansky (-glyceroPO4) Shenowara Jones (-glyceroPO4)

King- Armstrong (-nitrophenylPO4)

Bessy Lowry-Brock (-nitrophenylPO4)

Alkaline AminoTransferase (ALT) Serum Glutamic Pyruvic Transaminase (SGPT)

Found in the LIVER and RBCs More specific for liver than AST Marked elevation with viral hepatitis

Collection: AVOID HEMOLYSIS

 Catalyzes the reaction:

Alanine + ketoglutaric acid

ALT

Pyruvic Acid

Method:

Reitman Frankel
Addition of DNPH (2,4- dihydrophenylhydrazine) to pyruvic acid to produce color

ALT (Liver)
Substrate End Product
Location High Concentration Significance
Alanine - Keto

AST (Heart) Glutamate oxalacetate transaminase

Aspartate keto

Pyruvic Acid
Heart, LIVER LIVER

Oxaloacetic Acid
HEART, Liver HEART Mycocardial Infarction

Liver Disease

 ALT/AST RATIO de ritis ratio

Differentiates VIRAL from NON-VIRAL etiology
Viral Etiology: High ALT Non-viral: High AST If ALT/AST is >1 = VIRAL If ALT/AST is <1 = NON-VIRAL

Lactate DeHydrogenase (LD/LDH)

Catalyzes reversible lactate

pyruvate

using NAD+ as a cofactor

5 isoenzymes (electrophoresis):
LD1, LD2, LD3, LD4, LD5

 4 SUBUNITS in each Isoenzyme

ISOENZYME LD1 LD2 LD3 LD4 LD5 SUBUNITS HHHH HHHM HHMM HMMM MMMM SHORTHAND SUBUNITS LD H4 LD H3M LD H2M2 LD HM3 LD M4 HIGH CONCENTRATION Heart, RBC, Brain Heart, RBC, Brain

Brain, Kidney, Lung

Liver, Skeletal muscle, kidney Liver, Skeletal muscle, ileum

 Collection: AVOID HEMOLYSIS

clot must be separated from serum quickly to prevent increase in LD1 and LD2
Storage: 25C upto 24 hours Only LD4 and LD5 are utilized to test LIVER

DISORDERS. May also be elevated during skeletal muscle damage

Gamma-Glutamyl Transferase/Transpeptidase (GGT)

Increase in liver disease, metastatic

carcinoma , alcoholism and hepatobiliary obstruction

Marker for daily consumption of large

amount of alcohol or drugs (barbituates, phenytoin)

Method: SZAZ- substrate: gammaglutamyl

-nitroanilide

Pseudocholinesterase (ChE)
Cholinesterase (true and Pseudo)
Cleaves Acetylcholine: the bodys major

neurotransmitter True ChE

high activity in the CNS, RBC, lungs and spleen.
Pseudo-ChE or acylcholine acylhydrolase primarily produced in the liver Also produced by myocardium and pancreas Important in cleaving SUCCINYLCHOLINE

 Significant decrease seen after exposure to phosphorus compounds found in insecticide, nerve gases and pesticide.
Also seen in anesthetic poisoning. Substrate: acetylcholine Method:
Michael
Ellman

CARDIAC DISORDER PROFILE

CK-MB

AST
LD (LDH) 1 & 2 LD/HBD ratio MI Biomarkers

Creatine Kinase (CK)

found in cardiac, skeletal and brain muscle Has 3 isoenzymes:
CK-BB (CK1)= Brain

CK-MB (CK2)= Heart, muscle

CK-MM (CK3)= Muscle, heart

 Catalyzes

phosphocreatine + ADP
Methods:
Tanzer-Gilvarg: (forward)

creatine + ATP

Phospho Kinase + LD
Oliver- Rosalki: (reverse)

Lactate + NAD

Hexokinase + G6PD

6-P Gluconate + NADPH

ASpartate aminoTransferase (AST)

Most sensitive enzyme foe skeletal muscle

disease
Collection: AVOID HELOLYSIS Inhibited by all anticoagulants except heparin

(do not use ammonium heparin)

Lactate DeHydrogenase 1 & 2

LD 1 (Anodic), while LD 5 is (cathodic)

LD 1 and LD2 (HEAT STABLE)

LD5 (COLD LABILE) Used in evaluating Cardiac Disorders

 Normally LD1< LD2

Normal Ratio is 0.5-0.75 or <1 If LD1>LD2 it is called a flipped ratio
Flipped ratio is seen in MI (provided sample is not

hemolyzed) 50% MI: flipped ratio in 48hours 80% MI: flipped ratio in 72 hours

LD/HBD(hydroxybutiric dehydrogenase) Ratio

Normal LD/HBD ratio: 1.2- 1.6 If ratio is 0.8-1.2, MI is suspected

Non-Enzymatic MI Markers
MYOGLOBIN
Major protein responsible for oxygen supply of

striated muscle

TROPONIN
the troponin complex is a component of the thin

filament of striated muscle linked to actin Three Subunits:

Troponin I: an inhibitory subunit Troponin T: tropomyosin-binding subunit Troponin C: Calcium-binding subunit

ACUTE PANCREATITIS PROFILE

AMYLASE LIPASE

Amylase
found in the SALIVARY GLANDS and

PANCREAS
Breaks down starch to simple sugars Substrate: starch

starch/ amylum

AMS

maltose

Maltase

glucose

 Substrates:
Pancreatic AMS: diastase Salivary AMS: ptyalin

Serum AMS is usually pancreatic in origin

microAMS: unbound, free. 50,000 dal. AMS

found in urine macroAMS: bound to IgG, IgA. High MW. Measured in serum

 Methods
Saccharogenic
Measures amount of maltose produced (glucose: Somogyi)

Iodometric/amyloclastic

Measures starch remaining

Chromogenic

Measures dye released from breakdown of polysaccharide

Kinetic Method

Measures change of NAD to NADH at 340nm

Lipase
Breaksdown Triglyceride into fatty acids and

glycerol
Significance: Acute Pancreatitis Substrate: Olive Oil End Product: Fatty Acids Methods:
Cherry-Crandall Sigma-Tietz Titration

PROSTATIC CANCER PROFILE

ACP PSA

ACP (pACp)
Optimum pH: 5 Very Labile, Add 5M acetate buffer or citrate

tablet to preserve
ACP are found in Prostate, RBC, bone, liver,

kidneys, platelets
Substrate: organic Phosphate such as -glyceroPO4 and -nitrophenylPO4

 Method
Chemical Inhibition Test

If Total ACP is normal: Stop test If elevated: suggestive of prostatic CA do p-ACP by Chemical Inhibition Test
Cu++ RBC-ACP
p-ACP

Tartrate C4H4O6-2 Unaffected (-)

Inactivated (+)

Inactivated (+)
Unaffected (-)

PSA

(Prostate-Specific Antigen)

Member of the kallikrein family of serein

proteases uniquely produced form the epithelial cells of the prostate gland
Most useful TM for Prostate Cancer Drawback: weak in distinguishing prostate CA

from nonmalignant prostate lesions

Reference range: 0-4ng/mL

Good luck and God bless! Study well and Pray Harder!
-Maam Siv