title: Active site mapping studies of malate dehydrogenase : identification of essential amino acid residues by o-phthalaldehyde.
abstract: Mitochondrial malate dehydrogenase (MDH) was found to be rapidly inactivated by o-phthalaldehyde. MDH-o-phthalaldehyde adduct gives a characteristic absorption maximum at 337 nm. Moreover, this derivative shows fluorescence emission maxima at 405 nm when excited at 337 nm and 280 nm. These results were consistent with isoindole ring formation in which the -SH group of cysteine and epsilon-NH2 group of lysine participate in the reaction. The enzyme was found to be protected against o-phthalaldehyde inactivation by NADH, indicating that the essential residues are present at or near the coenzyme binding site. Stoichiometric results indicate that 4 isoindole derivatives are formed per enzyme molecule upon complete inactivation. However, 90% of the activity loss was accompanied by the formation of 2 moles of isoindole per mole of the enzyme. These approaches give consistent evidence that two cysteines along with two lysines in close proximity are essential for the enzymatic activity.
authors.affiliation: Department of Chemistry, Indian Institute of Technology, Kanpur.
authors.forename: S
authors.initials: S
authors.lastname: Sheikh
authors.forename: S S
authors.initials: SS
authors.lastname: Katiyar
mesh_terms: D003545:Cysteine / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D008929:Mitochondria, Heart / Q000201:enzymology; D011487:Protein Conformation; D009764:o-Phthalaldehyde / Q000494:pharmacology
chemical_list: D009764:o-Phthalaldehyde; D008291:Malate Dehydrogenase; D008239:Lysine; D003545:Cysteine