ENZYMOLOGY

ENZYMES

• Biologic proteins
• Catalyze biochemical reactions without altering the equilibrium point of the reaction or being
consumed or changed in composition
 DEINITION OF TERMS

• Active site
• Where the substance on which the enzyme acts (substrate)

• Allosteric site
• Bind regulator molecules

• Isoenzyme
• Different forms of enzymes based on electrophoretic mobility, solubility, or resistance to inactivation

• Isoform
• Results when an enzyme is subject to posttranslational modifications
• Cofactor
• Nonprotein molecule that may be necessary for enzyme activity
• Inorganic cofactors
• Chloride
• magnesium
• Coenzyme/organic cofactors
• NAD

• Prosthetic group
• Enzyme portion (Apoenzyme) + coenzyme (Prosthetic group)
• Forms a complete and active system, a holoenzyme

• Proenzyme or zymogen
• Inactive form of enzyme
 ENZYME CLASSIFICATION AND
NOMENCLATURE

• Systematic name
• Defining the substrate acted on
• The reaction catalyzed
• The name of any coenzyme involved in the reaction

• Recommended name
• When systematic name is too long

• EC numerical code containing four digits separated by decimal points

 FIRST DIGIT

• Six classes
1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyases
5. Isomerases
6. Liagases
FACTORS THAT INFLUENCE ENZYMATIC
REACTIONS
SUBSTRATE CONCENTRATION

• Enzyme–substrate (ES) complex

• The substrate readily binds to free enzyme at a low-substrate concentration
• First-order kinetics
• The substrate readily binds to free enzyme at a low-substrate concentration
• The reaction rate is directly proportional to substrate concentration
• Zero-order kinetics
• Free enzyme combines with excess free substrate
• The reaction rate depends only on enzyme concentration
 MICHAELIS-MENTEN CONSTANT (KM)

• Derived from the theory of Michaelis and Menten

• An expression of the relationship between the velocity of an enzymatic reaction and substrate
concentration
 PH

• Changes in pH may denature an enzyme or influence its ionic state

• Most physiologic enzymatic reactions occur in the pH range of 7.0 to 8.0
 TEMPERATURE

• Increasing temperature usually increases the rate of a chemical reaction

 COFACTORS

• Nonprotein entities that must bind to particular enzymes before a reaction occurs
• Inorganic cofactors
• Metallic (Ca2+,Fe2+, Mg2+,Mn2+, Zn2+, and K+)
• Nonmetallic (Br- and Cl–)

• Organic cofactors
• Nucleotide phosphates
• Vitamins
 INHIBITORS

• Interferes with the reaction

• Competitive inhibitors
• Physically bind to the active site of an enzyme

• Noncompetitive inhibitor
• Binds an enzyme at a place other than the active site

• Uncompetitive inhibition
• Inhibitor binds to the ES complex
• The enzyme–substrate–inhibitor
• complex does not yield product
 MEASUREMENT OF ENZYME ACTIVITY

• Activity is related to concentration

• Measures increase in product concentration photometrically
• Always performed in zero-order kinetics
 2 GENERAL METHODS IN MEASURING EA

• Fixed-time
• The reactants are combined, the reaction proceeds for a designated time, the reaction is stopped and a
measurement is made of the amount of reaction that has occurred.
• ^ rxn = ^ enzyme

• Continuous-monitoring or kinetic assay

• Multiple measurements, usually of absorbance change, are made during the reaction, either at specific time
intervals (usually every 30 or 60 seconds) or continuously by a continuous-recording spectrophotometer
• Advantageous over fixed-time methods
 CALCULATION OF ENZYME ACTIVITY

• Activity units
• The units used to report enzyme levels

• International unit(IU)
• The amount of enzyme that will catalyze the reaction of 1 mol of substrate per minute

• When enzymes are quantitated by measuring the increase or decrease of NADH at 340 nm, the
molar absorptivity (6.22 103 mol/L) of NADH is used to calculate enzyme activity.
 ENZYMES AS REAGENTS

• Enzymes may be used as reagents to measure many nonenzymatic constituents in serum

• Immobilized enzymes
• Chemically bonded to adsorbents, such as agarose or certain types of cellulose, by azide groups, diazo,
and triazine
ENZYMES OF CLINICAL SIGNIFICANCE
 CREATINE KINASE

• Molecular weight = approximately 82,000

• Predominant physiologic function occurs in muscle cells
• Storage of high-energy creatine phosphate

• Highest activities found in skeletal muscle, heart muscle, and brain tissue
• Smaller quantities in bladder, placenta, gastrointestinal tract, thyroid, uterus, kidney, lung, prostate,
spleen, liver, and pancreas
 DIAGNOSTIC SIGNIFICANCE

• Frequently elevated in disorders of cardiac and skeletal muscle

• Sensitive indicator of acute myocardial infarction (AMI) and muscular dystrophy (Duchenne
type)
• Duchenne type = 50 to 100 times the upper limit of normal (ULN)

• Vary with muscle mass

 THREE ISOENZYMES

• CK-BB (brain type)

• CK1

• CK-MB (hybrid type)

• CK 2

• CK-MM (muscle type)

• CK 3
 METHODS OF MEASUREMENT

• Electrophoresis
• Reference method
• Allowing visualization of adenylate kinase (AK) (hemolyzed)

• Ion-exchange chromatography
• Radioimmunoassay
• Immunoinhibition
 ASSAY ENZYME ACTIVITY

• Change in absorbance at 340 nm is determined

• Forward reaction is coupled with the pyruvate kinase–LDH–NADH
• pH 9.0
• Reverse reaction is coupled with the hexokinase–glucose-6-phosphate dehydrogenase–NADP
system
• pH 6.8
 SOURCE OF ERROR

• AK reacts with ADP to produce ATP

• Causing falsely elevated CK levels

• CK is inactivated by light
• Activity can be restored after storage in the dark at 4°C for 7 days or at -20°C for 1 month
when the assay is conducted using a sulfhydryl activator
 REFERENCE RANGE

• Total CK:
Male, 15–160 U/L (37°C)
Female, 15–130 U/L (37°C)
• CK-MB: < 6% total CK
 LACTATE DEHYDROGENASE

• Catalyzes the interconversion of lactic and pyruvic acids

• High activities are found in the heart, liver, skeletal muscle, kidney, and erythrocytes
• Lesser amounts are found in the lung, smooth muscle, and brain
 DIAGNOSTIC SIGNIFICANCE

• LDH is elevated in a variety of disorders

• Highest levels of total LDH = pernicious anemia and hemolytic disorders
• In AMI,
• Rise within 12 to 24 hours
• Peak levels within 48 to 72 hours
• May remain elevated for 10 days.
ISOENZYMES
ASSAY FOR ENZYME ACTIVITY
 SOURCE OF ERROR

• Any degree of hemolysis should render a sample unacceptable for analysis

• LDH activity is unstable in serum
• If the sample cannot be analyzed immediately, it should be stored at 25°C and analyzed within 48
hours
 REFERENCE RANGE

• LDH, 100–225 U/L (37°C)

 ASPARTATE AMINOTRANSFERASE

• Transferases
• Commonly referred to as a transaminase

• Serum glutamic-oxaloacetic transaminase (SGOT, or GOT)

 TISSUE SOURCE

• The highest concentrations are found in cardiac tissue, liver, and skeletal muscle
• Smaller amounts found in the kidney, pancreas, and erythrocytes.
 DIAGNOSTIC SIGNIFICANCE

• In AMI
• Rise within 6 to 8 hours
• Peak at 24 hours
• Return to normal within 5 days

• Elevations are frequently seen in pulmonary embolism

• Highest in acute hepatocellular disorders
• In viral hepatitis, levels may reach 100 times ULN
 ASSAY FOR ENZYME ACTIVITY

• AST are generally based on the principle of the Karmen method

• Coupled enzymatic reaction using malate dehydrogenase (MD) as the indicator reaction and monitors
the changein absorbance at 340 nm continuously as NADH is oxidized to NAD+

• pH 7.3 to 7.8
 SOURCE OF ERROR

• Hemolysis should be avoided because it can dramatically increase serum AST concentration
• Stable in serum for 3 to 4 days at refrigerated temperature
• Reference Range
• 5 to 30 U/L (37°C)
 ALANINE AMINOTRANSFERASE

• Catalyzes the transfer of an amino group from alanine to a-ketoglutarate with the formation of
glutamate and pyruvate
• Glutamic-pyruvic transaminase (SGPT, or GPT)
 TISSUE SOURCE

• High concentrations in the liver

• Considered the more liver-specific enzyme of the transferases
 DIAGNOSTIC SIGNIFICANCE

• Mainly to evaluation of hepatic disorders

• Remains normal in AMI
 ASSAY FOR ENZYME ACTIVITY

• Coupled enzymatic reaction using LDH as the indicator enzyme

• Change in absorbance at 340 nm measured
 SOURCE OF ERROR

• ALT is stable for 3 to 4 days at 4°C. It is relatively unaffected by hemolysis.

• Reference Range
• 6–37 U/L (37°C)
 ALKALINE PHOSPHATASE

• Catalyze the hydrolysis of various phosphomonoesters at an alkaline pH

• Functions to liberate inorganic phosphate from an organic phosphate ester
• Optimal pH for the reaction is 9.0 to 10.0
• The enzyme requires Mg2 as an activator
 TISSUE SOURCE

• Present on cell surfaces in most human tissue

• Highest concentrations are found in the intestine, liver, bone, spleen, placenta, and kidney
• In the liver, the enzyme is located on both sinusoidal and bile canalicular membranes
• Activity in bone is confined to the osteoblasts
• Production of bone matrix
 DIAGNOSTIC SIGNIFICANCE

• Evaluation of hepatobiliary and bone disorders

• Obstructive conditions > hepatocellular disorders
• Highest elevations in Paget’s disease (osteitis deformans)
 ISOENZYMES

• Liver
• Bone
• Intestine
• Placenta
 ASSAY FOR ENZYME ACTIVITY

• Calculation of ALP activity based on the molar absorptivity of p-nitrophenol

• p-Nitrophenylphosphate (colorless) is hydrolyzed to p-nitrophenol (yellow)
• Increase in absorbance at 405 nm
 SOURCE OF ERROR

• Hemolysis may cause slight elevations

• Reference Range
• 30 to 90 U/L (30°C)
 ACID PHOSPHATASE

• Same with ALP

• Optimal pH of 5.0
 TISSUE SOURCE

• Found in the prostate, bone, liver, spleen, kidney, erythrocytes, and platelets
 DIAGNOSTIC SIGNIFICANCE

• Historically, ACP measurement has been used as an aid in the detection of prostatic carcinoma,
particularly metastatic carcinoma of the prostate
ASSAY FOR ENZYME ACTIVITY
 SOURCE OF ERRROR

• Serum should be separated from the red cells as soon as the blood has clotted
• CP is stable for 2 days at room temperature.

• Reference Range
• Prostatic ACP 0 to 3.5 ng/mL
 Y-GLUTAMYLTRANSFERASE

• involved in:
• Peptide and protein synthesis
• Regulation of tissue glutathione levels
• Transport of amino acids across cell membranes
 TISSUE SOURCE

• Found primarily in tissue of the kidney, brain, prostate, pancreas, and liver
• Clinical applications are focused on evaluation of liver and biliary system disorders
 DIAGNOSTIC SIGNIFICANCE

• Elevated in virtually all hepatobiliary disorders

 ASSAY FOR ENZYME ACTIVITY

• p-nitroaniline at 405 to 420 nm

 SOURCE OF ERROR

• Stable for 1 week at 4°C

• No GGT in rbc

• Reference Range
• GGT: male, 6–45 U/L (37°C); female, 5–30 U/L (37°C)
 AMYLASE

• Catalyze the breakdown of starch and glycogen

 TISSUE SOURCE

• Acinar cells of the pancreas and the salivary glands

• Lesser concentrations are found in skeletal muscle and the small intestine and fallopian tubes
• AMS is the smallest enzyme, with a molecular weight of 50,000 to 55,000
 DIAGNOSTIC SIGNIFICANCE

• Diagnosis of acute pancreatitis

• In acute pancreatitis, serum AMS levels
• Rise at 2 to 12 hours after the onset of an attack
• Peak at 24 h
• Return to normal levels within 3 to 5 days

• Range from 250 to 1,000 Somogyi units per dL (2.55 ULN)

• Salivary gland lesions, such as mumps and Parotitis, and other intra-abdominal diseases, such as
perforated peptic ulcer, intestinal obstruction, cholecystitis, ruptured ectopic pregnancy,
mesenteric infarction, and acute appendicitis
 ISOENZYMES

• P isoamylase = Pancreatic tissue

• (P1, P2, P3)

• S isoamylase = Salivary gland tissue

• (S1, S2, S3)
 ASSAY FOR ENZYME ACTIVITY

• optimal pH is 6.9
• NADat 340 nm
 SOURCE OF ERROR

• Little loss of activity occurs at room temperature for 1 week or at 4°C for 2 months
• Morphine and other opiates = falsely elevated serum AMS levels

• Reference Range
• Serum, 25–130 U/L; urine, 1–15 U/h

• Conversion factor is 1.85

 LIPASE

• Hydrolyzes the ester linkages of fats to produce alcohols and fatty acids
 TISSUE SOURCE

• Found primarily in the pancreas

• Also present in the stomach and small intestine
 DIAGNOSTIC SIGNIFICANCE

• Confined almost exclusively to the diagnosis of acute pancreatitis

• More specific for pancreatic disorders than AMS
• LPS elevations persist for approximately 5 days in acute pancreatitis
 ASSAY FOR ENZYME ACTIVITY

• Cherry-Crandall method used an olive oil substrate

 SOURCE OF ERROR

• Negligible loss in activity at room temperature for 1 week or for 3 weeks at 4°C
• Hemoglobin inhibits the activity of serum LPS, causing falsely low values

• Reference Range
• 0–1.0 U/mL
 GLUCOSE-6-PHOSPHATE DEHYDROGENASE

• Catalyzes the oxidation of glucose-6-phosphate to 6-

phosphogluconate or the corresponding lactone
 TISSUE SOURCE

• Adrenal cortex, spleen, thymus, lymph nodes, lactating mammary gland, and erythrocytes
• Little activity is found in normal serum
 DIAGNOSTIC SIGNIFICANCE

• Functions to maintain NADPH in reduced form

• Regenerate sulfhydryl-containing proteins(glutathione)
• Protects hemoglobin from oxidation by agents that may be present in the cell
ASSAY FOR ENZYME ACTIVITY
 REFERENCE RANGE

• 10–15 U/g Hgb

 MACROENZYMES

• Serum enzymes that can be bound to either an immunoglobulin (macroenzyme type 1) or a

nonimmunoglobulin substance (macroenzyme type 2)
• Found in patients who have an unexplained persistent increase of enzyme concentrations in
serum
 DRUG-METABOLIZING ENZYMES

• Function primarily to transform xenobiotics into inactive, water-soluble compounds for

excretion through the kidneys
• Phase I reactions
• Catalyze addition or removal of functional groups through hydroxylation, oxidation, dealkylation,
dehydrogenation, reduction, deamination, and desulfuration reactions
• Often mediated by cytochrome P450 (CYP 450) enzymes
• Phase II reactions
• Xenobiotics transformed into more polar compounds through enzyme-mediated conjugation
reactions
• conjugated with glucuronide (UDP-glucuronyltransferase 1A1 [UGT1A1]), acetate (N-
acetyltransferase [NAT]), glutathione (glutathioneS-transferase [GST]), sulfate (sulfotransferase), and
methionine groups