Enzymes • Cofactor – non-protein molecule that may be required for an enzyme activity

• Biologic proteins that catalyze biochemical reactions 1. Coenzyme

• Appear in the serum or plasma (after tissue injury) • Organic cofactors
• Acts as a catalyst • Ex. NAD
o Increases the rate of particular chemical reaction 2. Activators
• Inorganic cofactors
– ++ ++
Functions of Enzymes • Ex. Cl , Mg , Zn
1. Physiologic functions 3. Metalloenzymes
• Hydration of CO2 • Inorganic ion attached to a molecule
• Nerve conduction
• Muscle contraction Notes:
• Nutrient degradation • When bound tightly to the enzyme, the coenzyme is called a prosthetic group
• Energy use • Apo enzyme (enzyme portion) and coenzyme forms complete and active system known
2. Diagnosis of diseases as holoenzyme
o Apoenzyme + prosthetic group = holoenzyme
General Properties • Digestive enzymes in its inactive form originally secreted from the organ of production is
Structure Description called a proenzyme or zymogen
Primary Contains amino acid sequence
Secondary Polypeptide chain twisting Denaturation
Tertiary Folds; results in structural cavities • Disruption of activity due to:
Quaternary Spatial relationship between the subunits (if enzyme contains more o Elevated temperature
than 1 polypeptide unit) § Low temperature – preserves enzyme activity
• Contains: o Extremes of pH
• Active site § Unfolding of enzyme structure
o Water-free cavity o Chemical addition
o Part of substrate on which the enzyme acts on
• Allosteric site Enzyme Theories
o Binds regular molecules 1. Emil Fisher’s / Lock and Key Theory
o Significant to the basic enzyme structure • Enzyme + substrate à enzyme-substrate complex
2. Kochland’s / Induced Fit Theory
• Isoenzyme • Substrate enters the active site of enzyme
o Different forms of the same enzyme capable of the same catalytic function in the • Enzyme clamps down around the substrate, forming an induced fit
body
o Represent enzymes from different genes that catalyze the same reaction Organ Specificity of Enzymes
o Ex. LDH-1 to -5
• Alloenzyme Level of
Enzyme Tissue / Organ
o Enzyme from different alleles of the same gene Specificity
o Differentiated based on electrophoretic mobility High ACP RBC and prostate
ALT Liver
Isoenzyme Isoforms AMS Salivary glands and pancreas
• Genetic origin Hybrid isoenzyme • Non-genetic origin LPS Pancreas
• Possess the ability to • Three hybrid isoenzyme • Multiple forms of Moderate AST Liver, heart, skeletal muscle
catalyze the enzyme’s • LD-2, LD-3, LD-4 enzyme post- CK Brain, heart, skeletal muscle
characteristic reaction translational ALP Liver, bone, kidneys
but that differ in Mixed dimer modification Low LD All tissues
structure • Mixed MB dimer of CK

• LD, CK, ALP

Enzyme Classification and Nomenclature o Point of saturation
• Standardized by Enzyme Commission of the International Union of Biochemistry
ex. Creatine kinase (CK), E.C. 2.7.3.2 • Enzyme reactions
st
1 : class (1-6 reactions) a. First-order kinetics
nd rd
2 , 3 : subclass, subsubclass (where enzyme is aligned) • Reaction rate is directly proportional to substrate concentration
th
4 : specific serial number
b. Zero-order kinetics
6 Reactions / Classes • Velocity of reaction is not affected by the addition of more substrate
1. Oxidoreductases – reduction reaction between two substrate at a certain point
2. Transferases – catalyze transfer of a group other than hydrogen from one substrate to • Reaction depends only on enzyme concentration
another • When maximum velocity is reached, the rate of increase in velocity
3. Hydrolases – catalyzes hydrolysis of various bonds is zero (zero order reaction)
4. Lyases – catalases removal of groups from substrates without hydrolysis; the production
contains double bonds • Lineweaver-Burk reaction
5. Isomerases – catalyze the interconversion of geometric, optical or positional isomers
6. Ligases – catalyze the joining of 2 substrate molecules, couples with breaking of the 1 𝐾# 1 1
pyrophosphate bond in ATP or similar compound = +
𝑉 𝑉#$% [𝑆] 𝑉#$%

Catalytic Mechanism of Enzymes o Km = negative reciprocal of the x-intercept

• Increased energy = increased reaction until it reaches the energy barrier o Used for more accurate determination of Vmax
• Activation energy o Helps in determination of types of enzyme inhibition
o The energy required to raise all molecules to the transition state in a chemical
reaction so that products may be formed 2. Enzyme concentration
• Enzyme level is high
Enzyme specificity o Faster reaction
a. Bond specificity o Reaction rate steadily increases as more substrate is added
b. Group specificity • The higher the enzyme concentration, the faster the reaction because more
c. Substrate specificity enzymes are present to bind with the substrate
d. Optical specificity
e. Geometrical specificity 3. Temperature
f. Cofactor specificity
°C
Factors that Influence Enzymatic Reaction 25, 30, 37 Enzymes are active
A. Factors Governing the Rate of Enzyme-Catalyzed Reaction 37 Optimum temperature for enzymatic reaction
1. Substrate concentration
40-50 Denaturation (significant)
• Michaelis & Menten, 1913
60-65 Inactivation of enzymes
o The substrate readily binds to free enzyme at a low substrate
Low temperature Enzymes are reversible inactive
concentration
Every 10°C increase Two-fold increase in enzyme activity
o With the amount of enzyme exceeding the amount of substrate, the
reaction rate steadily increases as more substrate is added
4. pH
o
• At optimal pH, the enzyme possess maximal activity
• Michaelis-Menten
• Most reactions occur in pH 7.0 – 8.0
𝑉#$% [𝑆]
𝑉= 5. Salt & protein concentration
𝐾# [𝑆]
• ↑ protein concentration = ↑ enzyme activity
o Km = concentration of substrate when reaction velocity is equal to half
the maximum velocity of the reaction
 6. Cofactors Major Clinical Enzymes
1. Aspartate Aminotransferase (E.C. 2.6.1.1)
Activator Coenzyme • Systematic name: L-Aspartate:2-oxaglutarate aminotransferase
• Inorganic cofactors • Organic cofactors • Old name: serum glutamic-oxaloacetic transaminase (SGOT or GOT)
• Modify spatial configuration of the • Acts as secondary substrate • Coenzyme: pyridoxal-5’-phosphate
;<=
enzyme • Coenzymes must always be provided in 𝐿– 𝑎𝑠𝑝𝑎𝑟𝑡𝑎𝑡𝑒 + ∝– 𝑘𝑒𝑡𝑜𝑔𝑙𝑢𝑡𝑎𝑟𝑎𝑡𝑒 𝑜𝑥𝑎𝑙𝑜𝑎𝑐𝑒𝑡𝑎𝑡𝑒 + 𝑔𝑙𝑢𝑡𝑎𝑚𝑎𝑡𝑒
• Link substrate to the enzyme or excess o Transfer of amino group between aspartate and ketoglutarate
coenzyme
• Tissue sources:
7. Anticoagulants o Cardiac tissue
o Liver
Anticoagulant Effect o Skeletal muscle
Heparin May inhibit AMS & ALT • Other sources:
Citrate Falsely lowers CK & ALP o Kidney
o Pancreas
8. Storage o RBCs

°C • 2 Isoenzyme Fractions:
–20 Preservation for longer period of time (enzyme) a. Cytoplasmic AST – predominant form in serum
2–8 Ideal for storage of substrate and coenzyme b. Mitochondrial AST – causes cellular necrosis
RT Ideal for storage of LD (LD-4 & LD-5)
• Reference values:
9. Inhibitors o 5 to 30 U/L (37°C)
a. Competitive inhibitor o Note: Hemolysis can cause a 10x increase in AST
• Inhibitor that competes with the substrate for binding to the active
catalytic site • Diagnostic significance:
Diagnostic Significance Level
b. Non-competitive inhibitor Hepatocellular disorder Very high / highest
• Inhibitor will bind to a site in the enzyme (allosteric) other than the active Viral hepatitis 100x ULN
site Cirrhosis Moderate / 4x ULN
Skeletal muscle disorders 4-8x ULN
c. Uncompetitive inhibitor Pulmonary embolism Elevated
• Inhibitor will bind to the enzyme-substrate complex, preventing o AST is not useful in diagnosis of acute myocardial infarction
dissociation o In congestive heart failure, AST increase
§ Reflecting liver involvement due to inadequate blood supply
Assay of Enzymes
• Enzyme activity is reported in IU (International Unit) • Method:
o To standardize reporting of enzyme activity a. Karmen method
o 1 IU = amount of enzyme that catalyzes conversion of 1 µmol of substrate to
product per minute ;<=
𝐿– 𝑎𝑠𝑝𝑎𝑟𝑡𝑎𝑡𝑒 + ∝– 𝑘𝑒𝑡𝑜𝑔𝑙𝑢𝑡𝑎𝑟𝑎𝑡𝑒 𝑜𝑥𝑎𝑙𝑜𝑎𝑐𝑒𝑡𝑎𝑡𝑒 + 𝑔𝑙𝑢𝑡𝑎𝑚𝑎𝑡𝑒
• Some enzyme activity results are reported in kat
o 1 kat = amount of enzyme that catalyzes conversion of 1 mol of substrate per
o Coupled enzymatic reaction:
second FG
o 1 kat = 16.7 nkat 𝑜𝑥𝑎𝑙𝑜𝑎𝑐𝑒𝑡𝑎𝑡𝑒 + 𝑁𝐴𝐷𝐻 + 𝐻 E 𝑚𝑎𝑙𝑎𝑡𝑒 + 𝑁𝐴𝐷 E

o Absorbance: 340 nm
o pH: 7.3 – 7.8
2. Alanine aminotransferase (E.C. 2.6.1.2) 3. γ-glutamyl transferase (2.3.2.2)
• Systematic name: L-Alanine:2-oxaglutarate aminotransferase • Systematic name: (5-Glutamyl) peptide: amino acid-5-glutamyl-transferase
• Old name: serum glutamic pyruvic transaminase (SGPT or GPT) • Common name: GGTP
• Coenzyme: pyridoxal-5’-phosphate • Standard abbreviation: GGT

• Tissue sources: • One of the large group of enzymes known as peptidase

o Liver o Catalyzes hydrolytic cleavage of peptides to form amino acids or smaller
o Kidney peptides
• Other sources: • Involved in:
o Pancreas, RBC, heart, skeletal muscle, lungs o Peptide and protein synthesis
o Regulation of tissue glutathione level
• Reference value: § Glutathione serves as γ-glutamyl donor
o 6 to 37 U/L (37°C) o Transports amino acids
• Location: canaliculi of hepatic cell; epithelial cell lining of the biliary duct
• Diagnostic Significance: • Tissue sources:
o Increased elevations are found in hepatocellular disorders that in extra- or o Kidney, brain, prostate, pancreas, liver
intrahepatic obstructive disorders • Reference values:
• Method o Male: 6-45 U/L (37°C)
a. Karmen method o Female: 5-30 U/L (37°C)
;J=
𝐿– 𝑎𝑙𝑎𝑛𝑖𝑛𝑒+ ∝– 𝑘𝑒𝑡𝑜𝑔𝑙𝑢𝑡𝑎𝑟𝑎𝑡𝑒 𝑝𝑦𝑟𝑢𝑣𝑎𝑡𝑒 + 𝑔𝑙𝑢𝑡𝑎𝑚𝑎𝑡𝑒 OP=
JG
𝑝𝑦𝑟𝑢𝑣𝑎𝑡𝑒 + 𝑁𝐴𝐷𝐻 + 𝐻 E
𝑙𝑎𝑐𝑡𝑎𝑡𝑒 + 𝑁𝐴𝐷 E 𝐿– 𝛾– 𝑔𝑙𝑢𝑡𝑎𝑚𝑦𝑙 – 𝑝– 𝑛𝑖𝑡𝑟𝑜𝑎𝑛𝑖𝑙𝑖𝑑𝑒 + 𝑔𝑙𝑦𝑐𝑦𝑙𝑔𝑙𝑦𝑐𝑖𝑛𝑒 𝑝– 𝑛𝑖𝑡𝑟𝑜𝑎𝑛𝑖𝑙𝑖𝑛𝑒 +
o Absorbance: 340 nm 𝛾– 𝑔𝑙𝑢𝑡𝑎𝑚𝑦𝑙 𝑔𝑙𝑦𝑐𝑦𝑙𝑔𝑙𝑦𝑐𝑖𝑛𝑒
o pH: 7.3 – 7.8
• Diagnostic significance
AST ALT o GGT is confined mainly to the evaluation of liver & biliary system disorders
Major organ Heart Liver Condition / Disorder GGT level
Substrate Aspartic α-ketoglutaric acid Alanine α-ketoglutaric acid Hepatocellular disorder Elevated
End product Glutamic acid + Glutamic acid + Biliary tract obstruction Higher elevation
oxaloacetic acid pyruvic acid Infectious hepatitis 2-5x ULN
Color developer Sulfanilic acid 2,4 DNPH Chronic alcoholism 2-3x ULN
Color intensifier HCl 0.4 NaOH Patients receiving enzyme-inducing drugs 4x ULN
Method Doumas Biggs Reitman & Frankel Acute pancreatitis 5-15x ULN
Diabetes mellitus Elevated
Increased transaminases: Myocardial infarction Elevated
• ALT: Moderate smokers 5x ULN
o Toxic hepatitis Heavy smokers 20x ULN
o Wolff-Parkinson White syndrome Patients taking oral contraceptives Decrease by 20%
st
o Chronic alcoholism 1 trimester of pregnancy Decrease by 25%
o Hepatic cancer
o Reye’s syndrome • Methods:
o Viral hepatitis a. Kinetic-photometric method (Szasz)
• ALT activity is higher than AST except in: • Temperature: 30°C
o Alcoholic hepatitis b. Colorimetric endpoint method (Rosalki)
o Cirrhosis • Temperature: 37°C
o Liver neoplasia
• DeRitis ratio – ratio of ALT to AST • Enzyme activity:
o > 1 – ratio in acute hepatitis o γ-glutamyl-p-nitroanilide: substrate
 o glycylgycine: acceptor § Healing of bone fractures
o γ-glutamyl glycylglycine: transferred product § Pregnancy
o p-nitroaniline: donor residue • Approximately 2-3x ULN
• Detected within 16-20 weeks of pregnancy
o Absorbance: 405-420 nm • Also increase in complications:
o End color: yellow o Hypertension
o pH: 8.2 o Eclampsia
o Increased threatened abortion
4. Alkaline Phosphatase (E.C. 3.1.3.1) § Presence of intestinal ALP isoenzymes
• Systematic name: Orthophosphoric monoester phosphohydrolase (alkaline • Blood group B and O; and secretors (Ag in body fluids)
optimum) • Note: Blood group A: ALP may increase after fatty meal intake
• Common abbreviation: ALP
• Standard abbreviation: ALP o Decreased ALP:
§ After blood transfusion or cardiopulmonary bypass (transiently
• Non-specific enzyme decreased)
• Liberates inorganic phosphate from an organic phosphate ester § Hypophosphatasia
§ Zinc deficiency
;JT
𝑝ℎ𝑜𝑠𝑝ℎ𝑜𝑚𝑜𝑛𝑜𝑒𝑠𝑡𝑒𝑟 + 𝐻R 𝑂 𝑎𝑙𝑐𝑜ℎ𝑜𝑙 + 𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒
o pH: 9 – 10 Major Tissue Source Isoenzyme Inhibitor
o Activators: Mg, Mn, Co Liver Liver ALP Levamisole reagent
o Cofactors: Zn Bone Bone ALP Levamisole reagent;
3M urea
• Tissue source: Placenta Placental ALP Phenylalanine reagent
o Intestine Intestine Intestinal ALP Phenylalanine reagent
o Liver: in sinusoidal & bile canalicular membrane
o Bone: osteoblast o Bone ALP – heat-labile
o Spleen o Electrophoresis – most effective method in determining ALP isoenzymes
o Placenta § Liver fraction: most anodal: migrates the fastest
o Kidney
• Reference value: • Enzyme activity
o 30 – 90 U/L (37°C) o Liver and bone ALP will not separate in untreated serum
o Neuramidase
• Diagnostic Significance: § Separates bone & liver ALP
§ Serum treated for 15 mins at 37°C
Disease / Condition ALP level § To remove terminal cyanic acid residues
Bile tract obstruction 3-10x ULN
Hepatocellular disorders (hepatitis, cirrhosis) <3x ULN; slightly increased Carcinoplacental ALP
Point of Differentiation
Bone disorders: Regan ALP Nagao ALP
• Paget’s disease (osteitis deformans) 20-25x ULN Conditions Carcinomas: lung, breast, Adenocarcinoma of the
• Osteomalacia 2-4x ULN ovarian, colon pancreas and bile duct
• Rickets 2-4x ULN Heat stable Yes Yes
Resisting deterioration Yes Yes
• Hyperparathyroidism Slight to moderate elevation
(65°C for 30 min)
• Osteoporosis Slightly increased
Inhibitor Phenylalanine Phenylalanine
L-leucine
o Normally elevated:
§ Period of growth
§ Geriatric
 • ALP Methods

Method Substrate End Products

Bodansky β-glycerol phosphate Inorganic phosphate +
Shinowara glycerol
Jones
Reinhart
King & Armstrong Phenyl phosphate Phenol
Bessy, Lowry & Brock P-nitrophenol phosphate P-nitrophenol /
Bowers & McComb Yellow nitrophenoxide ion
Huggins & Talalay Phenolphthalein Phenolphthalein red
diphosphate
Moss α-naphthol phosphate α-naphthol
Klein, Babson & Read Buffered phenolphthalein Free phenolphthalein
phosphate

• Measurement of ALP Activity

o Bowers & McComb (Continuous Monitoring Technique)
§ Modified Szasz
§ Most recommended

4– 𝑛𝑖𝑡𝑟𝑜𝑝ℎ𝑒𝑛𝑦𝑙 𝑝ℎ𝑜𝑠𝑝ℎ𝑎𝑡𝑒 + 𝐻R 𝑂
;JT FVWX [\$[[$]V\^ $_ $`a bc
4– 𝑛𝑖𝑡𝑟𝑜𝑝ℎ𝑒𝑛𝑜𝑥𝑖𝑑𝑒 𝑏𝑒𝑛𝑧𝑜𝑖𝑑 4– 𝑛𝑖𝑡𝑟𝑜𝑝ℎ𝑒𝑛𝑜𝑥𝑖𝑑𝑒 (𝑞𝑢𝑖𝑛𝑜𝑛𝑜𝑖𝑑)

• Benzoid form – colorless

• Quinonoid form – yellow

§ Absorbance: 405 nm
§ pH: 10.3

5.