ENZYMES GENERAL CLASSIFICATIONS

● Enzymes are specific proteins that catalyze 1. Oxidoreductases

biochemical reactions without altering the equilibrium ● Removal or addition of electron
point of the reaction or being consumed or changed in ● REDOX reaction (Reduction-Oxidation)
composition. Example:
● Measured in terms of activity ● Dehydrogenase - Lactate dehydrogenase (LDH),
● appear in the serum after cellular injury, or sometimes Malate dehydrogenase (MDH), Isocitrate
from degraded cells, in smaller amounts form storage dehydrogenase (ICD)
areas ● Oxidase - cytochrome oxidase
● Abnormal large amounts of enzyme in serum evident
of organ damage 2. Transferase
● Catalyze the trnasfer of chemical group from one
Holoenzymes substrate to another
● Combination of coenzyme and apoenzymes Example:
Apoenzymes ● Aspartate aminotransferase (AST)
● Protein portion subject to denaturation in which ● Alanine aminotransferase (ALT)
enzymes loss its activity ● Creatine kinase (CK)
Isoenzyme ● Creatinine phosohokinase (CPK)
● Present in individual with similar enzyme activity ● Gamma glutamyl transferase (GGT)
● Differ in the physical and biochemical and ● Ornithine carbonyl transferase (OCT)
immunologic characteristics
Metalloenzyme 3. Hydrolase
● Enzyme whose metal ions are intrinsically part the ● Hydrolyse the splitting of a bond by the addtion of
molecule such as catalases and cytochrome oxidases water
Proenzyme Example:
● Inactive precursor of enzymes ● Esterase
● Aka ZYMOGENS ○ Acid phosphatase (ACP)
○ Alkaline phosphatase (ALP)
Substrate ○ Cholinesterase (CLS)
● Substances acted upon by the enzymes which are ○ Lipase (LPS)
specific for each particular enzyme ● Peptidases
Cofactor ○ Trypsin (PTS)
● Non protein substances compound ○ Pepsin (PPS)
○ Leucine aminopeptidase (LAP)
Nomenclature ● Glycosidase
PRACTICAL / TRIVIAL NAME ○ Amylase
“ase” ○ Amylo 1,6 glycosidases
● Lipase: enzymes on lipids ○ Galactosidase
● Protease: enzymes on proteins
Transferase 4. Lyases
- treansfer of amino acids ● Remove groups from substrate without hydrolysis
Kinase ● Leaving only double bonds in the molecular structure
- kinetic / transfer to phosphatase for high energy of the product
Phosphatase Example:
- Effect of hydrolysis on phosphate esters ● Aldolae
Dehydrogenase ● Glutamate decarboxylase
- removal of hydrogen atoms from substrates ● Pyruvate decarboxylase
● Tryptophan decarboxylase
SYSTEMATIC NAME
● According to numerical designation given by enzyme 5. Isomerases
commision (E.C) ● Catalyzes the intramolecular rearragement of the
○ E.C.1.1.1.7 - Lactate Dehydrogenase substrate compounds
○ E.C.3.2.1.1 - Amylase Example:
○ E.C.2.6.1.2 - Alanine aminotransferase ● Glucose phosphate isomerase
● 1st number - define CLASS ● Ribose phosphate isomerase
● Next 2 numbers - indicate SUBCLASS
● Last number - specific SERIAL NUMBER 6. Ligases (Syntheses)
● Joins two substrate molecule together using the
energy released from hydrolyzing pyrophosphate
bond to high energy phosphate bond
COFACTOR KOSHLAND’S INDUCED FIT THEORY
● Coenzyme ● Based on the attachment of a substrate to the active
○ Organic molecule site of an enzyme, which then cause conformational
○ Hastens enzymatic reaction but undergo changes in the enzyme
change or is consumes to another products ● MORE ACCEPTABLE
○ Ex.: NAD / NADPH ● ENZYME is the one that will ADJUST
● Activator
○ Metal ion Types of reaction order
○ Serves as bridge to hold substrate and 1. Zero order reaction
enzyme together ● Rate of reaction linear with time independent of
○ The primary catalytic center condentration of substrate
○ Stabilizing agent in the conformation for ● Directly proportional to enzyme concentration
catalytic activity 2. First order reaction
○ Ex.: Amylase, Cl, Br-, LDH, Zn2, Lipase, ● Rate of reaction is determined by the concentration of
Ca++ the substrate

ENZYME KINETICS Factors Affecting Enzyme Reaction

● Enzyme catalyses a reaction by combining with its Enzyme concentration
substrate to create E-S complex (ES) ● Increase in the concentration of enzyme produces an
● According to Michaelis & Menten, E-S complex can increase in the rate on reaction, provided that the
either dissociate back to E + S or breakdown to P and other condition remain the same and that a constant
free enzyme but excess amount of substrate present

Substrate concentration
● An increase in the concentation of substrate produces
also an increase on the rate of reaction. Provided all
other conditions are kept constant
Michaelis-Menten Equation ● However, the rate of the reaction reaches a maximal
● Gives the means to determine the total enzyme values at a particular concentration of substrate and
concentration in serum and other body fluids higher concentration of substrate do not result in
higher rate of reaction (Saturation kinetics)

Temperature
● Rate of any reaction increase 2-3 times for every
● V is measured velocity of reaction 10deg C temperature (Q10)
● Vmax is maximum velocity ● 25degC, 30degC, 37degC - enzymes are ACTIVE
● [S] is substrate concentration ● 60-65degC - INACTIVATION of enzymes
● Km is Michaelis-Menten constant of enzyme for a
specific substrate. Hydrogen Ion concentration or pH
● 7-8 pH (Alkaline) optimum
Types of specificity ● Enzymatic reaction proced at other fastest rate at an
A. Absolute specificity optimum pH and are considerably slowed or even
● Combines with only one substrate and catalyzes only stopped at higher/lower pH values
one corresponding reaction
B. Group soecificity Competitive inhibitor
● Enzymes combining with all substrates containing a ● These are substances that compete with substrate for
particular _____ group enzyme binding because theyare chemically
C. Bond specificity analogous to substrate bind to active sitesif enzymes
● Enzymes are specific to chemical bonds
D. Stereoisometric specificity Non competitive inhibitor
● Enzyme that predominantly combine with only one ● Do not resemble the substrate and bind to the
particular isomeror a certain compound enzyme in areas other than active sites (Usually metal
ions)
EMIL FISHER’S LOCK AND KEY theory
● On rigid enzyme molecule into which substrate fits Uncompetitive inhibition
● Key (substrate) Lock (enzyme) ● Inhibits enzyme by binding to enzyme substrate
● the shape of the key (S) must conform into the lock complex
(E)
Enzyme induction SPECIFIC ENZYMES
● This phenomenon states that a certain enzyme has Phosphatases
ability to adapt to their biochemical enzymes ● Characterized by its ability to hydrolyze a large variety
of organic phosphatase esters with formation of an
Types of enzyme assays alcohol and phosphate ion
1. Endpoint Analysis ● Hydrolases
● Reaction is initiated by addition of substrate ● Class 3 (Classification of enzymes
● Reaction allowed to procees for period of time
● Measurement is done at the end of reaction Alkaline Phosphatase (E.C 3.1.3.1 / ALP)
● Disadvantage: Underestimation of TRUE enzyme ● Alkaline orthosohoric monoester phosphohydrolase
activity and linearity cannot be observed ● Reference value: 30-90 uL
● ALP is an enzyme involved in cleavage of lhosohate
2. Multipoint and Kinetic Assay containing compounds in alkaline pH. Facilitates
● Change on concentration of the indicator substance at across substances across cell membranes
several intervals ● Several isoenzymes exist which include
● Continuous measurement of change in concentrate ○ Placental Isoenzyme
as function of time ○ Intestinal Isoenzyme
Use of coupled reaction ○ Liver Isoenzyme
● Enzymatic activity measured by coupling activity with ○ Bone Isoenzyme
colorimetric reaction Liver ALP
● inhibit LEVAMISOLE
Unit for expreassing enzyme activity Bone ALP
International Unit (I.U or U) ● most heat labile
● equivalent to the amount of enzyme that catalyzes the ● Inhibit UREA and LEVAMISOLE
conversion of 1 micromole of substrate per minute Placental ALP
under controlled condition ● Most heat labile
● 1 I.U = 16.7 Katal Unit ● inhibit PHENYLALANINE rgt.
● 1 KU = 0.06 u/L Intestine ALP
Katal Unit ● Inhibit PHENYLALANINE rgt.
● Equivalent to the amount of enzymes that catalyzes
the conversion of 1 mole of substrate per second Carcinoolacental ALP
under controlled conditions Regan ALP
● Found in lung, breast, ovarian, gynecological cancers
Pitfall in enzyme assay ● Bone ALP co-migrator
● Hemolysis cause falsely elevated values due to ● Most heat stable
release of enzymes from RBC ● Inhibit PHENYLALANINE rgt.
● Serum rather than plasma is preferred specimen due Nagao ALP
to adverse effects of anticoagulants on enzyme ● adenocarcinoma of pancrease and bile duct, pleural
activity cancer, variant of regan
● Lactescent or milky serum causes variable absorption ● Inhibited by L-LEUCINE and PHENYLALANINE
by spectrophotometer Kasahara ALP
● Storage ● hepatoma / helatocellular carcinoma

Storage of samples Methods of determination

● Most enzymes stable at 6degC, 24 hrs ● Bowers and McComb (continuous monitoring
● Few enzymes are inactivated at Ref temp (LD4 and 5) technique) pH 10-15, 405nm
● LDH (Room temp) ● p-nitrophenylphosphatase —> p-nitrophenol +
phosphate ion
QC program for Enzymes Assay ● Most specific method
● Strict adherence to Zero Order Kinetics
● Proportionally with increments of sample Measurement
● Use of pooled frozen serum or stable reference ● use p-nitrophenyl phosphate substrate at an alkaline
materials as control pH
● Replicate measurent to evaluate precision of assay ● Enzyme: Zn, Mg, other cations
● Chelates (EDTA, oxalate, citrate) falsely lower activity
● Activity of enzyme increase slightly on storage due to
loss of inhibitors
● ALP relatively stable at 4degC for 1 week
● Optimum pH 8.6-10
Increased ALP CHEMICAL INHIBITION TECHNIQUE
● Osteomalacia
RBC ACP Prostatic ACP
● Rickets
● Bone cancer Copper + -
● Sprue
● Hyperthyroidism Tartrate - +
● Hepatitis and Cirrhosis
● Copper Resistant: Prostatic ACP
Decreased ALP
● Tartrate Resistant: RBC ACP
● After blood transfusion / cardiolulmonary bypases
(transiently decrease)
ACP elevation
● Malnutrition
Moderate elevation of total ACP
● Hypophosphate ia (prolonged severe low levels)
● Female breast CA
● Zinc deficiency (Zinc is necessary cofactor ALP
● Paget’s disease
activity)
● Hyperthyroidism
Acid phosphatase ( E.C. 3.1.2.3)
Non-prostatic ACP elevation
● Catalyzes same reaction by ALP
● Niemann-pick disease
● Active at pH 5.0
● Gaucher’s disease
● hydrolytic enzyme se reted by number of cells
● Myelocytic leukemia
● Several isoenzyme of ACP each with tissue specificity
● Isoenzyme may be fractined into 5 bands
Diagnostic significance
● Detection of prostatic carcinoma
Tissue sources
● Prostate (major source)
● RBC, platelets, bone
Reference value
● 2.5 - 11.7 uL (total ACP male)
● 0-3.5 ng/mL (prostatic ACP)

ACP isoenzyme
Band 1
● Prostate gland (major source)
● Inhibited by TARTRATE
Band 2
● form granulocyte
Band 3
● Major form present in plasma
● Derived from platelets, erythrocytes, and monocytes
Band 5
● Found in osteoclasts
● Resistant to TARTRATE inhibition

Tartrate - INHIBITED ACP (Prostatic isoenzyme)

● Prostatic cancer
● Benign prostatic hyperplasia
● Proststic infarction
● Urinary tract obstruction,carcinoid tumors of rectum,
prostatice massage
● Medico-legal cases (ACP has implications suspected
rape
○ Positive (+) ACP in vaginal swabs if semen
is present for first 12 hrs upto 4 days

Tartrate - RESISTANT ACP (Bone isoenzyme)

● Active osteoclast-mediated bone resorption
● Gaucher’s cell
● Hairy cell leukemia