ENZYMES

ENZYMOLOGY- Study of enzymes 3. HYDROLASES

– Activity of enzymes • Hydrolysis of various bonds
– Chemical reactions it catalyze • Addition of water to a bond resulting in bond breakage
– Clinical use – E.C. 3.2.1.1 : 1,4-D-Glucan Glucanohydrolase
ENZYMES- Biologic catalysts • Alpha-Amylase
- Hasten chemical reactions
- Not consumed during the reactions
- Not undergoing a chemical change after the
reactions.

4. LYASES
• Addition of a group to a double bond or the removal of a
group to form a double bond
– Carbonic Anhydrase
– Citrate Lyase

NOMENCLATURE OF ENZYMES
1. Substrate + -ase
5. ISOMERASES
 Lipid = lipase
• Rearrange the functional groups within a molecule and
 Ester = esterase catalyze the conversion of one isomer into another
 Protein = protease – Phosphoglycerate mutase
2. Reaction it catalyzes
 Oxidation = oxidase
 Reduction = reductase
 Hydrolysis = hydrolase
 Dehydrogenase = remove hydrogen atoms,
transferring them to a coenzyme
 Decarboxylase = remove carboxyl groups
3. Enzyme Commission Nomenclature (E.C.)
6. LIGASES
– E.C. 1.1.1.21
• Catalyze a reaction in which C-C, C-S, C-O, or C-N bond is made or
– 1st digit = class
broken
– 2nd digit = subclass
• Accompanied by an ATP-ADP interconversion
– 3rd and 4th = serial number
CLASSIFICATION OF ENYZYMES
1. Oxidoreductases 2. Transferases
3. Hydrolases 4. Lyases
5. Isomerases 6. Ligases

1. OXIDOREDUCTASES
• Oxidation and reduction
 Oxidation – removal of H ion TERMS
 Reduction – accept H ion
• Substrate – Acted upon by the enzyme
 E.C. 1.1.1.27 : L-lactate NAD+Oxidoreductase
– Specific
 Lactate Dehydrogenase
• Isoenzyme – Different form but with the same
action
• Cofactor – Non-protein molecule
• Activator – Inorganic cofactor
• Coenzyme – Organic cofactor
• Apoenzyme – Polypeptide portion
2. TRANSFERASES
• Holoenzyme – Apoenzyme + Coenzyme
• Transfer of functional groups other than hydrogen from one
substrate to another • Proenyzme – Also known as zymogen
– E.C. 2.6.1.1 : – Inactive form of enzyme
L-Aspartate: 2- Oxaloglutarate Aminotransferase – It is then converted, usually by
• Aspartate Aminotransferase proteolysis, to the active form when it
has reached the site of its activity
 FACTORS THAT INFLUENCE ENZYMATIC REACTIONS
1. Substrate Concentration
• First-order Reactions
- Are those which proceed at a rate exactly
proportional to the concentration of one reactant
ENZYME KINETICS
AP
• Michaelis – Menten Theory Rate of reaction is exactly proportional to the rate of
E + S  E-S disappearance of A or the appearance of P
• Second-order Reactions
- Are those in which the rate is proportional to the
product of the concentration of two reactants
A+BP
Rate of reaction is exactly proportional to the rate of
disappearance of A or the appearance of P
• Zero-order Reactions
- The reactions are zero-order with respect to the
reactants
- Rate of reaction depends on the concentration of
catalysts or on some factor other than the
concentration of the molecular species undergoing
reaction

2. Enzyme Concentration
– The higher the enzyme level, the faster the reaction will
proceed
LOCK AND KEY THEORY 3. pH
– pH = 7.0 – 8.0
– Changes in pH may denature the enzyme
– Protein in nature
4. Temperature
– 37 degrees Celsius (normal)
– Denaturation at 40-50 degrees Celsius
– assay temperatures:
• At 25, 30, or 37 degrees C
5. Cofactors
– Nonprotein entities that must bind to particular enzymes
before a reaction occurs
– Metallic or nonmetallic
– Calcium, ferrous, magnesium, manganese, zinc, potassium
INDUCED FIT THEORY ions
• Activators
– Proper substrate binding
– Linking substrate to the enzyme or coenzyme
– Undergoing oxidation or reduction
6. Inhibitors
– Interfere with enzyme reactions
• Competitive
• Noncompetitive
• Uncompetitive
 Continuous-Monitoring Method (Kinetic Assay)
– Multiple measurements
– Absorbance change
 Increasing in absorbance
 Decreasing in absorbance
– Time intervals

CALCULATION OF ENZYME ACTIVITY

International Unit (IU)
– Amount of enzyme that will catalyze the reaction of 1
micromole of substrate per minute under specified conditions
International Unit per Liter (IU/L)
– Enzyme concentration
Katal Unit (mole/s)
– Amount of enzyme that will catalyze the reaction of 1 mole of
substrate per second under specified conditions
- 1.0 IU = 16.7 nkat
ENZYMES OF CLINICAL SIGNIFICANCE
AMYLASE (AMS)
 E.C. 3.2.1.1
 1,4-D-Glucan Glucanohydrolase
 Breakdown of starch and glycogen to monosaccharides
 Calcium ions and chloride- Activators

Tissue Sources
 Acinar cells of the pancreas
- Pancreatic AMS
 Salivary glands
- Salivary AMS
- Ptyalin
Isoenzymes
 P-type isoenzymes
- Pancreas
 S-type isoamylase
- Salivary gland
- Lungs
- Fallopian tubes
 Both are metalloenzymes – containing calcium ion
 Bromide and iodide serve as activators (Henry)

Poperties
 MW 50,000 – 55,000
MEASUREMENT OF ENZYME ACTIVITY  Readily filtered by the renal glomerulus (appears in the
• Increase in product concentration urine)
• Decrease in substrate concentration  Mouth – starch initial digestion by salivary AMS,
• Decrease or increase in coenzyme concentration Inactivated in the stomach
 Small intestine – final digestion by pancreatic AMS
• An increase in the concentration of the altered enzyme
Diagnostic Significance
 Salivary gland involvement
 Acute Pancreatitis
 Rise: 6-48 hours after onset of an attack (Henry); 2-
12 hours (Bishop and Hubbard)
 Peak: 24 hours (Bishop and Hubbard)
 Normalize: 3-5 days (Bishop and Henry)
 Mumps
ENZYMATIC ASSAYS  Parotitis/Parotiditis
Coupled-Enzyme Assay  Perforated peptic ulcer
– Substances other than substrate or coenzyme are necessary  Intestinal obstruction
and must be present in excess – NAD+ or NADH  Cholecystitis
Fixed-Time Method (End-point)  Ruptured ectopic pregnancy
– Reactants are combined  Mesenteric infarction
– Reaction proceeds for a designated time  Acute appendicitis
– Reaction is stopped  Macroamylasemia
– Measurement is made of the amount of reaction has - Results when the AMS molecule combines with
occurred immunoglobulins to form a complex that is too large
to be filtered across the glomerulus
Assay for Enzyme Activity
 Amyloclastic
- AMS is allowed to act on a starch substrate to which
iodine has been attached
- As AMS hydrolyzes the starch, the iodine is released
and a decrease in the initial dark-blue color intensity
of the starch-iodine complex occurs
- THE DECREASE IN COLOR IS PROPORTIONAL TO THE
AMS CONCENTRATION
 Saccharogenic
- Uses a starch substrate that is hydrolyzed by the
action of AMS to its constituent carbohydrate
molecules that have reducing properties
- THE AMOUNT OF REDUCING SUGARS IS THEN
MEASURED WHERE THE CONCENTRATION IS
PROPORTIONAL TO AMS ACTIVITY
- Classic reference method in Somogyi units
 Chromogenic
- Uses a starch substrate to which a chromogenic dye
has been attached, forming an insoluble dye-
substrate complex
- As AMS hydrolyzes the starch substrate, smaller dye-
substrate fragments are produced, and these are
water-soluble
- THE INCREASE IN COLOR INTENSITY OF THE SOLUBLE
DYE-SUBSTRATE SOLUTION IS PROPORTIONAL TO AMS
ACTIVITY
 Coupled-Enzyme
- Have been used to determine AMS activity by a
continuous-monitoring technique in which the
change in absorbance of NAD+ at 340 nm is
measured
- Optimum pH 6.9
Sources of Error
 Morphine and other opiates
- Falsely elevate serum AMS levels
- Can cause constriction of Oddi’s sphincter and
pancreatic ducts
- With consequent elevation of inarticulate pressure
causing regurgitation of AMS into the serum
 Citrate or oxalate as anticoagulant
- Falsely low activity
- AMS is calcium-containing enzyme
- Heparin does not interfere with its activity
 Contamination of saliva
- Approx. 700 times that of serum
- Red cells contain no AMS, so hemolysis generally
presents no problem with most methods except
those coupled-enzyme methods
LIPASE (LPS)
 E.C. 3.1.1.3
 Triacylglycerol Acylhydrolase
 Hydrolyzes the ester linkages of fats to produce alcohols
and fatty acids
 Catalyzes the partial hydrolysis of dietary TAG in the
intestine to the 2-monoglyceride and fatty acids
 Enzymatic activity is specific for the fatty acid residues at
positions 1 and 3 of the TAG molecules
 Substrate must be an emulsion for activity to occur
 Reaction rate is accelerated by the presence of colipase
and bile salt
 Proteins, bile acids, and phospholipids
- Inhibit serum lipase
 Colipases
- Reverse this inhibition
 Calcium is necessary for maximal lipase activity
 Heavy metals and quinine inhibit LPS activity
Tissue Sources
 Primarily in the pancreas
 Also present in the stomach and small intestine
Diagnostic Significance
 Almost exclusively to the diagnosis of acute pancreatitis
 In Acute Pancreatitis
- Rise: 2-12 hours after onset of an attack
- Peak: 24 hours
- Persist for approximately 5 days (Bishop); for about 7-
10 days (Henry)
- Extent of LPS elevations does not correlate with
severity of the disease
 Found also in other intra-abdominal conditions
- Duodenal ulcers, perforated peptic ulcers, intestinal
obstruction, acute cholecystitis
 Normal in conditions of salivary gland involvement
Assay for Enzyme Activity
 Includes estimation of liberated fatty acids and
turbidimetric methods
 Classic Cherry-Crandall Method
 Substrate: Olive Oil
 Measured the liberated fatty acids by titration
after a 24- hour incubation
 Turbidimetric Methods
 As the fats are hydrolyzed by LPS, the particles
disperse, and the rate of clearing can be
measured as an estimation of LPS activity
 Colorimetric Methods
 Based on coupled reactions with enzymes such
as peroxidase or glycerol kinase
Sources of Error
 LPS is stable in serum, with negligible loss in activity at RT
for 1 week or for 3 weeks at 4 degrees Celsius
 Hemolysis should be avoided because hemoglobin
inhibits serum LPS
 ASPARTATE AMINOTRANSFERASE (AST)
 E.C. 2.6.1.1
 L-Aspartate: 2-oxaloglutarate Aminotransferase
 Transfer of an amino group between aspartate and
alpha-keto acids to form oxaloacetate and glutamate
- Former name: serum glutamic-oxaloacetic
transaminase (SGOT or GOT)
- Pyridoxal phosphate(Vit. B6) COFACTOR
ALANINE AMINOTRANSFERASE (ALT)
 E.C. 2.6.1.2
 L-Alanine: 2-oxaloglutarate Aminotransferase
 Transfer of an amino group between alanine and alpha-
keto acids to form glutamate and pyruvate
- Former name: serum glutamic-pyruvic transaminase
(SGPT or GPT)
- Pyridoxal phosphate – coenzyme

Tissue Sources
 Transamination reaction is important in intermediary  Widely distributed in human tissue
metabolism because of its function in the synthesis and  Liver – highest concentration
degradation of amino acids  Considered the more liver-specific enzyme of the
 Ketoacids formed are ultimately oxidized by the transferase
tricarboxylic acid cycle to provide a source of energy Diagnostic Significance
 Confined mainly to evaluation of hepatic disorders
Tissue Sources - Higher elevations are found in hepatocellular
 Widely distributed in human tissue disorders than in extrahepatic or intrahepatic
 Cardiac tissue – highest concentration obstructive disorders
 Liver - In acute inflammatory conditions of the liver, ALT is
 Skeletal muscles higher than AST
 Kidney
 Pancreas Assay
 Erythrocytes  Consists of a coupled enzymatic reaction using LD as the
Isoenzyme Fractons indicator enzyme
 Cell cytoplasm - Catalyzes the reduction of pyruvate to lactate with
- Predominant from occurring in serum simultaneously oxidation of NADH (340 nm)
 Mitochondria - Optimal pH: 7.3 – 7.8
- Increases in disorders producing cellular necrosis Sources of Error
Diagnostic Significance  ALT is stable for 3 – 4 days at 4 degrees Celsius
 Limited mainly to the evaluation of hepatocellular  Relatively unaffected by hemolysis
disorders and skeletal muscle involvement
 AMI ALKALINE PHOSPHATASE (ALP)
- Rise: 6 – 8 hours  E.C. 3.1.3.1
- Peak: 24 hour  Orthophosphoric Monoester Phosphohydrolase (Alkaline
- Normalized: within 5 days – Not useful in the Optimum)
diagnosis of AM - Catalyze the hydrolysis of various
 Pulmonary embolism phosphomonoesters at an alkaline pH
 CHF - Liberate inorganic phosphate from an organic
 Acute Hepatocellular Disorders phosphate ester with the concomitant production of
 Viral Hepatitis an alcohol
 Liver cirrhosis
 Muscular Dystrophies
 Inflammatory conditions
Assay for Enzyme Activity
 Generally based on Karmen Method
- Incorporates a coupled enzymatic reaction using
malate dehydrogenase (MD) as the indicator  Optimal pH: 9.0 – 10.0
reaction and monitors the change in absorbance at - Varies with the substrate used
340 nm continuously as NADH is oxidized to NAD+  Magnesium and Zinc ions  ACTIVATORS
- Optimal pH: 7.3 – 7.8 Tissue Sources
 Reaction with dinitrophenylhydrazine couples color  Present on cell surfaces in most tissues
reagent with keto acid product  Intestine
 Reaction with diazonium salts couples the salt with the  Liver – Sinusoidal and bile canalicular
keto acid product and forms a color membranes
 Bone (Osteoblasts – involved in production of
bone matrix)
 Spleen
 Placenta
 Kidney
Isoenzymes (Electrphoresis)
Sources of Error  Liver ALP – migrates the fastest
 Hemolysis should be avoided  Bone ALP
 AST activity is stable in serum for 3 – 4 days at refrigerated  Placental ALP
temperatures  Intestinal ALP
 2 Fractions of Liver ALP  Bone Disorders
Major liver fraction - Paget’s Disease (osteitis deformans)
- Hepatobiliary conditions - Osteomalacia
Fast liver fraction - Rickets
- Alpha1 liver - Hyperparathyroidism
- Metastatic carcinoma of the liver and - Osteogenic carcinoma
hepatobiliary diseases - Healing bone fractures
- As a valuable indicator of obstructive - During periods of physiologic bone growth
liver disease  Normal Pregnancy
 Bone ALP - Increased ALP
- Increases due to osteoblastic activity - Average approximately 1 ½ times ULN
- Normally elevated in children during periods of - Can be detected between weeks 16 and 20
growth and in adults older than age 50 - Persists until the onset of labor
 Intestinal ALP - Activity returns to normal within 3 – 6 days
- Depends on the blood group and secretor status of - May be elevated in hypertension, preeclampsia,
the individual (“B” and “O” ) and eclampsia; threathened abortion
- Bound by erythrocytes of “A” group  Decreased ALP
- Increases after consumption of fatty meal - Inherited condition of hypophosphatasia
- Diseases of the digestive tract and cirrhosis - Inadequate bone calcification
Isoenzymes (Heat Stability) Assay for Enzyme Activity
 ALP activity is measured before and after heating the  Continuous-Monitoring technique based on a method by
serum at 56 degrees Celsius for 10 minutes Bowers and McComb
- If the residual activity before heating is less than 20% - Calculation of ALP activity based on the molar
of the total activity before heating – Bone ALP absorptivity of p-nitrophenol
- Greater than 20% - Liver ALP - p-nitrophenylphosphate (colorless) is hydrolyzed to
p-nitrophenol (yellow)
 Placental ALP - Increases in ABS at 405 nm is directly porportional to
- Most heat stable ALP activity
- Resist heat denaturation at 65 degrees Celsius for 30 - Bessy-Lowry-Brock Method
minutes
 Intestinal ALP
 Liver ALP
 Bone ALP
Isoenzymes (Chemical Inhibition)
 Phenylalanine
- Inhibits intestinal ALP and placental ALP to a much
greater extent than liver and bone ALP
- Impossible to differentiate the four fractions
 Levamisole Sources of Error
- Inhibits bone ALP and liver ALP (Henry)  Hemolysis can cause slight elevations because ALP is 6
Isoenzymes (Abnormal Factions Associted with Neoplasm) times more concentrated in RBC than in serum
 Regan Isoenzyme  Should be run ASAP
- Has been characterized as an example of an - Activity increases 3 – 10% on standing at RT or 4
ectopic production of an enzyme by malignant degrees Celsius for several hours
tissue  Diet may induced elevations “B” and “O” individuals who
- Detected in various carcinomas are secretors
- Lung, breast, ovarian (highest incidence), colon - 25% higher following fat meal
- Migrates as the bone ALP
- Most heat stable of all ALP
- Resist denaturation (65 degrees Celsius for 30
minutes)
- Inhibited by phenylalanine
 Nagao Isoenzyme
- Carcinoplacental Alkaline Phosphatases
- Similarities to the placental isoenzyme
- 3% to 15% in cancer patients
- Considered as a variant of Regan isoenzyme
- Its electrophoretic, heat-stability, and phenylalanine-
inhibition properties are identical to the Regan
fraction
- Can also be inhibited by L-leucine
- Detected in metastatic CA of pleural surfaces and
adenocarcinoma of the pancreas and bile duct
Diagnostic Significance
 Biliary Tract Obstruction
- ALP levels range from 3 – 10 times the upper limit of
normal
- Primarily as a result of increased synthesis of the
enzyme induced by cholestasis
 Hepatocellular Disorders
- Hepatitis and cirrhosis
- Usually less than 3 times ULN