Biochemistry II - Seminars Doc. RNDr. Jiří Dostál, CSc. Department of Biochemistry, Fac. Med., MU Brno jrdostal@med.muni.cz Literature * J. Tomandl, E. Táborská: Biochemistry -- Seminars II, MU Brno, 1996 selected chapters, see the syllabus Enzymes in Clinical Biochemistry Seminar No. 1 - Chapter 4 - You are supposed to know ... * Enzymes -- main features, properties * Coenzymes -- structures, functions * Enzyme kinetics * Enzyme activity Isoenzymes -- General Features * Genetically determined differences in primary structure * Catalyze the same reaction * May have different subcellular distribution (cytoplasm × mitochondria) * May have different tissue distribution * May be combined from more subunits (quarternary structure) * May differ in kinetic properties (K[M]) * Usually are determined by electrophoresis Q. 2 (p. 27) Explain the terms: proenzyme, isoenzyme, isoform A. 2 * Proenzyme (zymogen) -- inactive form of enzyme that becomes active after partial proteolysis example: pepsinogen (r) pepsin * Isoenzyme -- see previous page * Isoform -- more general term, includes true isoenzymes and pseudoisoenzymes (posttranslational variations) Lactate dehydrogenase (LD) * Tetramer * Two different chains (H - heart, M - muscle) * Five isoenzymes: LD[1] (H[4]), LD[2] (H[3]M), LD[3] (H[2]M[2]), LD[4] (HM[3]), LD[5] (M[4]) * Widely distributed in body * Total activity determination -- nonspecific finding * LD[1] + LD[2] ..... marker of myocardial infarction (MI) * Today is LD assay considered out-of-date Creatine kinase (CK) * Dimer * Two different chains (M -- muscle, B -- brain) * Three isoenzymes: MM (muscle), MB (heart), BB (brain) * Major isoenzyme in blood is MM (95 %) * MB form in blood: 0 -- 6 % * BB in blood: traces (BB cannot pass across blood-brain barrier) * MB isoenzyme .... excellent marker of myocardial infarction Enzymes in Blood Enzymes in Blood Q. 6 Why are low activities of cellular enzymes detected even in serum of healthy people? A. 6 Low activities of intracelular enzymes in extracelular fluid (blood plasma, serum) are the consequence of physiological cell disintegration. Main Tissue Distribution of Enzymes Intracellular Location of Enzymes Consider the AST/ALT ratio * AST/ALT > 1 ......... severe liver damage * AST/ALT < 1 ......... mild liver damage Enzymes of Clinical Significance Catalytic concentration of some enzymes Q. 7 What enzymes might appear in blood *In mild hepatocellular damage b) In serious hepatocellular damage A. 7 Q. 8 Write equations of reactions catalyzed by: ALT AST LD ALT Reaction alanine + 2-oxoglutarate D ALT Reaction alanine + 2-oxoglutarate D pyruvate + glutamate AST reaction aspartate + 2-oxoglutarate D AST reaction aspartate + 2-oxoglutarate D oxaloacetate + glutamate LD reaction lactate + NAD^+ D LD reaction lactate + NAD^+ D pyruvate + NADH + H^+ Q. 9 The levels of most blood enzymes are increased in newborns and infants. What enzyme persists elevated till puberty? A. 9 ALP -- the bone isoenzyme activity persists till puberty Biochemical Diagnostic of MI