CLINICAL CHEMISTRY LABORATORY

ENZYME (biocatalysts)
Introduction
• protein molecules that catalyze biochemical reactions
• participate in virtually all cell function
• location: within the cell
• increased concentration = cell injury
• enzymes in circulation = no function
• discovered by BUCHNER (1900) while fermenting
yeasts
• name means “in yeasts”
• Induced Fit Model – Kochland
SGPT- enzyme used to evaluating liver function (0-40
MIg/ML)

- NO CALIBRATION FOR ENZYME

2 types CC quantitative analysis

ENDPOINT METHOD-allowing the entire reaction to take
place before sample will be measured the quantity.
(product of reaction is what measured)

KINETIC METHOD – measure rate of analyte reagent

complex formation. (rate of reaction) COFACTOR (enhance enzymatic reaction/activity)
Reagent plus sample (for 5 mins.) • nonprotein entity
• essential for enzyme catalytic activity
Substrate + Enzyme = Product • ACTIVATORS (inorganic electrolytes, calcium) and
COENZYMES (organic, NADP)
Properties & Terminologies
• ACTIVE SITE • APOENZYME
• water-free cavity • inactive enzyme - cofactor
• where substrate binds
• dictates enzyme specificity • HOLOENZYME
• active enzyme + cofactor
• ALLOSTERIC SITE
• may bind regulator molecules • PROSTHETIC GROUP
• active enzyme + coenzyme (organic cofactor)
• ENZYME-SUBSTRATE COMPLEX
• Lock and Key Model - Emil Fisher • ISOENZYME
• different enzymes catalyzing the same reaction
• separated through electrophoresis
• migrates fastest – isoenzyme 1

Enzyme Classification
• classified according to:
• biochemical function
• indicating substrate
• class of reaction catalyzed
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• NOTE: SCIENTIFIC NAME
• 1 st – substrate
• 2 nd – reaction
• 3 rd – coenzyme

FIRST DIGIT - class

1. OXIDOREDUCTASES – oxidation-reduction FIRST-ORDER KINETIC
2. TRANSFERASES – transfer of function group • double substrate = double enzymatic activity
3. HYDROLASES – hydrolysis reactions
4. LYASES – group elimination to form double bonds SECOND-ORDER KINETIC
5. ISOMERASES – isomerization • double substrate = quadruple enzymatic activity
6. LIGASES – bond formation coupled with ATP
hydrolysis ZERO-ORDER KINETIC
• amount of substrate has no effect on enzymatic
activity
SECOND DIGIT
• subclass ENZYME CONCENTRATION
• the higher the enzyme level, the faster the reaction will
THIRD DIGIT proceed
• sub-subclass
COFACTOR
FOURTH DIGIT • ACTIVATORS
• serial # specific to each enzyme • metallic or nonmetallic
• Ca2+, Fe2+, Mg2+, Zn2+
REDUCTION- receive • COENZYMES
OXIDATION- give • heme, biotin, FAD, CoA, vitamins

Examples INHIBITORS
• Glucose-6-phosphate Dehydrogenase Competitive (bind in active site)
• E.C. 1.1.1.49 • inhibitor binds at the same site as the
• Gamma-Glutamyl Transferase (seen in alcoholism. substrate
Severe case: Cirrhosis) • Ex. Dihydrofolate reductase is inhibited by
In liver organ transplantation: Need HLA (Human methotrexate
Leukocyte Antigen) type
• E.C. 2.3.2.2 Noncompetitive (bind at allosteric site)
• Angiotensin-Converting Enzyme • inhibitor binds at a site distinct from the
• E.C. 4.1.2.13 substrate-binding site

Factors Affecting Enzyme Reactions Uncompetitive (wait substrate to bind to

• SUBSTRATE CONCENTRATION implement inhibition)
• 1913: Michaelis-Menten Hypothesis • inhibitor binds to ES complex
• rate of reaction = substrate concentration

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Enzymes are measured in terms of:
• change in substrate concentration
• change in the product concentration
• change in coenzyme concentration

2-ways of measuring enzymes

FIXED-TIME
• reactants are combined
• reaction proceeds for a designated
time
TEMPERATURE • reaction is stopped
• increased temperature = increased chemical • measure amount of reaction that
reaction occurred
• 60-65C : inactivation of enzymes
• Ref/Freezing Temp: inactivates enzymes CONTINUOUS MONITORING/KINETIC METHOD
• Repeated Freezing and thawing: enzyme • multiple measurements
denaturation • specific time interval: 30 or 60 seconds
• PREFERRED
pH
• 7.0-8.0 Units of Expressing Enzymatic Activity

STORAGE INTERNATIONAL UNIT (IU or U)

• -20C: for long period • micromole substrate/minute
• 2-8C: ideal for substrate and coenzymes
• Room Temp: LDH storage KATAL UNIT (KU)
• 1 mole of substrate/second
HEMOLYSIS
• increased enzyme concentration Causes of Increased Enzyme Levels
• LACTESCENCE • Impaired removal of enzyme from plasma
• decreases enzyme concentration • Tissue necrosis and degeneration
• Increased permeability of cell membrane
SALT and PROTEIN CONCENTRATION • Increase in the # of cells or cell production
• increased ionic strength = decreased
enzyme activity MAJOR CLINICAL ENZYMES
• increased protein concentration =
increased enzyme activity Alkaline Phosphatase
• a.k.a. Alkaline Orthophosphoric Monoester
ANTICOAGULANTS Phosphohydrolase • EC 3.1.3.1
• Heparin: best • reaction:
• Citrate: falsely lowers CK and ALP • Organic Phosphate + ALP = Inorganic Phosphate +
Alcohol
EDTA: falsely increases concentration except • Optimal pH for reaction: 9.0-10.0
Renin • Activator: Magnesium • major tissue sources:
• liver, bone, placenta, intestine
al, renal, spleen
• Reference Value: • 30-90 U/L
Enzyme Activity
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ALP: Diagnostic Significance • measure ALP before and after heating the serum at
56C for 10 minutes • imprecise
• Hepatobiliary Disorders • depends on many factors:
• predominant in obstructive conditions: • temperature control
• in Hepatitis and Cirrhosis: 3-10x ULN • timing
• method sensitivity
Bone Disorders: involving the osteoblasts • Procedure:
• highest in Paget’s disease (osteitis deformans) • heat at 56C for 10-15 minutes

• other bone disorders asso. with increased ALP Selective chemical inhibition
• osteomalacia • uses phenylalanine
• rickets • inhibits intestinal and placental ALP greater
• hyperparathyroidism than liver and bone
• osteogenic sarcoma • 3M urea
• also increased in healing bone fractures and • inhibits bone ALP
periods of physiologic bone growth • Levamisole
• normal pregnancy: 1.5x ULN • inhibits liver and bone ALP
• 16th-20th week of gestation until the onset of
labor Carcinoplacental ALP
• increased n hypertension, preeclampsia,
eclampsia, and threatened abortion Regan ALP
• Decreased ALP • lung, breast, ovarian, colon, gynecological
• inherited hypophosphatasia cancers
• absence of bone isoenzyme • co-migrates with bone ALP
• most heat stable ALP
ALP Isoenzymes • inhibited by phenylalanine
• LIVER ALP – most anodal • useful in monitoring prognosis
• BONE ALP
• PLACENTAL ALP Nagao ALP
• INTESTINAL ALP – least anodal • adenocarcinoma of the pancreas and bile duct,
pleural cancer
ELECTROPHORESIS • variant of Regan
most useful single technique for ALP isoenzyme analysis • inhibited by L-leucine and phenylalanine

Liver ALP: METHODS

• divided into 2 fractions: Bowers and McComb (continuous monitoring technique)
• major liver band • pH 10.15
• fast liver or alpha1 liver smaller fraction • 405 nm
• P-nitrophenylphosphate + ALP = p-nitrophenol
Bone ALP: Direct Immunochemical Method + phosphate ion
• increased in osteoblastic activity • other methods: relative substrate
• elevated in children during periods of growth nonspecificity
and in adults older than 50 years old
• Intestinal ALP
• depends on blood group and secretor status

Heat Inactivation
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