Chemical modification of NADP-isocitrate dehydrogenase from Cephalosporium acremonium evidence of essential histidine and lysine groups at the active site.
NADP-isocitrate dehydrogenase from Cephalosporium acremonium CW-19 has been inactivated by diethyl pyrocarbonate following a first-order process giving a second-order rate constant of 3.0 m-1. s-1 at pH 6.5 and 25 degrees C. The pH-inactivation rate data indicated the participation of a group with a pK value of 6.9. Quantifying the increase in absorbance at 240 nm showed that six histidine residues per subunit were modified during total inactivation, only one of which was essential for catalysis, and substrate protection analysis would seem to indicate its location at the substrate binding site. The enzyme was not inactivated by 5, 5'-dithiobis(2-nitrobenzoate), N-ethylmaleimide or iodoacetate, which would point to the absence of an essential reactive cysteine residue at the active site. Pyridoxal 5'-phosphate reversibly inactivated the enzyme at pH 7.7 and 5 degrees C, with enzyme activity declining to an equilibrium value within 15 min. The remaining activity depended on the modifier concentration up to about 2 mm. The kinetic analysis of inactivation and reactivation rate data is consistent with a reversible two-step inactivation mechanism with formation of a noncovalent enzyme-pyridoxal 5'-phosphate complex prior to Schiff base formation with a probable lysyl residue of the enzyme. The analysis of substrate protection shows the essential residue(s) to be at the active site of the enzyme and probably to be involved in catalysis.
Olano
J
J
Departmento de Bioquímica y Biología Molecular, Universidad de León Spain.
Soler
J
J
Busto
F
F
De Arriaga
D
D
D000164:Acremonium / Q000201:enzymology*; D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004793:Enzyme Reactivators / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry*; D019811:Hydroxylamine / Q000494:pharmacology; D007521:Isocitrate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D011732:Pyridoxal Phosphate / Q000494:pharmacology
D004791:Enzyme Inhibitors; D004793:Enzyme Reactivators; D019811:Hydroxylamine; D006639:Histidine; D011732:Pyridoxal Phosphate; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+); D008239:Lysine; D004047:Diethyl Pyrocarbonate