ENZYMOLOGY.pdf

CLINICAL CHEMISTRY 2 LECTURE/ENZYMOLOGY ENZYMOLOGY ENZYMES GENERAL PROPERTIES ENZYME CLASSIFICATION AND NOMENCLATURE Enzymes are specific biological proteins OXIDOREDUCTASES Catalyze the removal or that catalyze biochemical reactions...

CLINICAL CHEMISTRY 2 LECTURE/ENZYMOLOGY ENZYMOLOGY ENZYMES GENERAL PROPERTIES ENZYME CLASSIFICATION AND NOMENCLATURE Enzymes are specific biological proteins OXIDOREDUCTASES Catalyze the removal or that catalyze biochemical reactions addition of electrons (redox without altering the equilibrium point or reaction). being consumed. TRANSFERASES Catalyze the transfer of a Found in all body tissues, especially in chemical group other than serum after cellular injury. hydrogen from one Composed of specific amino acid substrate to another. sequences. HYDROLASES Catalyze hydrolysis or splitting of a bond by the ENZYME addition of water (hydrolytic STRUCTURES reactions). PRIMARY Specific amino Acid LYASES Catalyze removal of groups STRUCTURE sequence. from substrates without SECONDARY Twisting of polypeptide hydrolysis. The product STRUCTURE chains. contains double bonds. TERTIARY Folding due to structural ISOMERASES Catalyze the intramolecular STRUCTURE cavities. arrangement of the substa. QUATERNARY Arrangement of more LIGASES Join two molecules with STRUCTURE than one polypeptide covalent bonds. unit, referring to the ❖ IUB- System assigns name and code to each spatial relationship enzyme. between subunits. *INTERNATIONAL UNION of BIOCHEMISTRY FORM OF ENZYME ISOENZYMES Different forms of an EC. CODE NO. enzyme that catalyze the OXIDOREDUCTASES 1.1.1.27 same reaction. 1=class (oxidoreductase) ISOFORM Variants resulting from 1= subclass of oxidoreductase post-transitional 1= subclass of the subclass of the oxidoreductases modifications. 27 =lactate dehydrogenase COFACTOR Non-protein molecules necessary for enzyme activity. VELOCITY VMAX x(s)/ Km + S ACTIVATOR Inorganic cofactors (CL, S Substrate and Mg ions, Calcium, V Velocity Zinc, K, Iron, Mn) Km Michael-Menten Inorganic compounds. constant COENZYME Organic cofactor (NAD) Organic compounds. SAPLAD, JOHNA MAY C. 1  CLINICAL CHEMISTRY 2 LECTURE/ENZYMOLOGY ENZYME KINETICS TYPES OF INHIBITORS Catalytic Activation energy COMPETITIVE INHIBITORS Inhibitor that competes Mechanism of Enzymes catalyze (reversible/irreversible) with substrate for the enzyme: reactions by (gahaman) active site lowering activation NON-COMPETITIVE Inhibitor that binds to a energy level. INHIBITORS different site, altering ENZYME SPECIFICITY (irreversible) enzyme function ABSOLUTE Specific to only 1 substrate (marites) GROUP Specific to all substrates of a UNCOMPETITIVE Inhibitor binds only to chemical group INHIBITORS the enzyme substrate BOND Specific to chemical bonds (reversible) complex STEREOISOMETRIC Specific to 1 optical isomer (sumisingit/epal) of a compound. FACTORS THAT INFLUENCE ENZYMATIC REACTIONS CLINICALLY SIGNIFICANCE ENZYMES SUBSTATE Changes can lead to CONCENTRATION denaturation or altered ENZYMES ONSET PEAK ELEVATION enzyme activity. (H) (H) (days) ENZYME Substrate concentration CK 4-8 12-24 3-4 CONCENTRATION affects reaction rate until CK-MB 4-8 24-38 2-3 saturation point is reached AST 8-12 24 5 PH It is the optimum for a rate LDH 12-24 72 10 factor that influences our enzymatic reaction. Any CREATININE Enzyme that catalyzes the changes in pH will cause KINASE (CK) conversion of creatine and uses denaturation of enzyme. ATP to form phosphocreatine 7.0-8.0 and ADP. TEMPERATURE The rate of denaturation (found in muscle tissue, brain, increases as the and heart) temperature increases. CLINICAL Elevated CK, especially CK-MB, COFACTORS can be an activator or SIGNIFICANCE is significant in diagnosing coenzyme. These are our myocardial infarction and inorganic cofactors (calcium, muscle disorders. k, iron, manganese, zinc) ASSAY ENZYME Catalyzes both forward & activators (Cl, Mg Ions) ACTIVITY reverse reactions involving enzyme. phosphorylation of creatinine INHIBITORS Enzymatic reactions may not or ADP. progress with the reaction. SOURCE OF ERROR Hemolysis, serum must be stored in a dark place, because it is inactivated by sunlight. REFERENCE RANGE Male: 46-171 U/L (37C) Female: 34-145 U/L (37) SAPLAD, JOHNA MAY C. 2  CLINICAL CHEMISTRY 2 LECTURE/ENZYMOLOGY ALANINE Catalyzes the transfer of LACTATE DEHYDROGENASE Catalyzes the conversion of AMINOTRANSFERASE amino group from alanine to (LDH) lactate to pyruvate, using (ALT) alpha-ketoglutarate, NAD+ as a cofactor (found producing pyruvate and in nearly all tissues, with glutamate. (Primarily found in high concentration in the the liver with smaller heart, liver, kidneys, and amounts in the kidneys, skeletal muscle). heart, and skeletal muscle) CLINICAL SIGNIFICANCE Elevated LDH levels can CLINICAL Hepatic disorder indicate tissue damage or SIGNIFICANCE disease, such as myocardial ASSAY FOR ENZYME Coupled enzymatic reaction infarction, liver disease, and ACTIVITY using lactate dehydrogenase certain cancers. Isoenzyme as indicator enzyme, which patterns help localize tissue catalyzes reduction of damage. pyruvate to lactate with ASSAY FOR ENZYME Catalyzes interconversion of simultaneous oxidation of ACTIVITY lactic & pyruvic acids using NADH. coenzyme NAD, in either SOURCE OF ERROR Stable for 3-4 days at 4C; forward or reverse relatively unaffected by direction. hemolysis SOURCE OF ERROR Hemolysis, temperature REFERENCE RANGE 7-45 U/L (37C) REFERENCE RANGE 125-220 U/L (37C) ASPARTATE Catalyzes the transfer of an ALKALINE PHOSPATASE Catalyzes the hydrolysis of AMINOTRANSFERA amino group from aspartate to (ALP) phosphate esters, resulting SE (AST) alpha-ketoglutarate, producing in the release of inorganic oxaloacetate and glutamate. phosphate. (Found in liver, (found in liver, heart, skeletal bones, kidneys, intestine, muscle, kidneys, and brain) spleen, CLINICAL Hepatocellular disorders (viral CLINICAL SIGNIFICANCE Hepatobiliary (biliary tract SIGNIFICANCE hepatitis, cirrhosis) skeletal obstruction) & bone) muscle disorders (muscle Paget’s disease, dystrophies, inflammatory ostomalacia, rickets, conditions), pulmonary hyperparathyroidism embolism ASSAY FOR ENZYME Various methodologies are ASSAY FOR ENZYME Based on Karmen method ACTIVITY used, including a ACTIVITY continuous-monitoring SOURCE OF ERROR Hemolysis, stable serum for 3-4 technique (Bowers & days at refrigerated temps. MaComb) REFERENCE RANGE 5-35 U/L (37C) SOURCE OF ERROR Hemolysis, run as soon as possible, high fat meal. REFERENCE RANGE 42-128 U/L (30C) (M/F 20- 50 Y/o) SAPLAD, JOHNA MAY C. 3  CLINICAL CHEMISTRY 2 LECTURE/ENZYMOLOGY AMYLASE Catalyzes the hydrolysis of ACID PHOSPATASE Catalyzes the hydrolysis of starch and glycogen into sugars, (ACP) phosphate esters at acidic pH, primarily maltose and glucose. releasing inorganic phosphate. (acinar cells of pancreas & (prostate, bone, liver, spleen, salivary gland) kidney, erythrocytes, platelets) CLINICAL Acute pancreatitis, disorders CLINICAL Prostatic carcinoma, SIGNIFICANCE causing salivary gland lesions SIGNIFICANCE hyperplasia of prostate, (mumps, parotitis), prostatic surgery, osteoclasts, intraabdominal diseases Paget’s disease, breast cancer ASSAY FOR 4 main approaches: amyloclast, with bone metastases, ENZYME ACTIVITY saccharogenic chromogenic, Gaucher’s disease continuous monitoring ASSAY FOR Same techniques as in alkaline SOURCE OF ERROR Stable for 1 week at room ENZYME ACTIVITY phosphatase, except performed temp. & 2 months at 4C; in an acid Ph. REFERENCE RANGE Serum 28-100 U/L; Urine: 1-15 SOURCE OF ERROR Serum should be separated U/hour from red cells as soon as blood clotted; serum should be used GAMMA- Catalyze the transfer of immediately, frozen or GLUTAMYL gamma-glutamyl groups from acidified. TRANSFERASE(GGT) peptides to amino acids or REFERENCE RANGE Prostatic ACP: 0-3.5 ng/ml other peptides. (kidney, brain, prostate, pancreas, liver) CLINICAL Hepatobiliary disorders (biliary SIGNIFICANCE tract obstruction), hepatic LIPASE Catalyzes the hydrolysis of parenchyma, alcoholism, acute tryglycerides into free fatty pancreatitis, diabetes mellitus, acids and glycerol. (pancreas; myocardial infarction also, in stomach & small ASSAY FOR ENZYME y-glutamyl-p-nitroanilide is intestines) ACTIVITY most widely accepted CLINICAL Acute pancreatitis, other substrate used in GGT analysis; SIGNIFICANCE intraabdominal diseases y-glutamyl residue is (penetrating duodenal ulcers, tranxferred to glycylglycine, perforated peptic ulcers, releasing p-nitroaniline intestinal obstruction, acute SOURCE OF ERROR Stable for 1 week at 4c; cholecystitis) hemolysis not concern ASSAY FOR Estimation of liberated fatty REFERENCE RANGE Male: 6-55 U/L (37C); Female: ENZYME ACTIVITY acids (triolein is substrate), 5-38 U/L (37C) turbidimetric methods, colorimetric methods SOURCE OF ERROR Hemolysis REFERENCE RANGE