Chemical modification of NADP-isocitrate dehydrogenase from Cephalosporiumacremonium - 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 givin g 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 inactiv ation, only one of which was essential for catalysis, and substrate protect ion analysis would seem to indicate its location at the substrate binding s ite. The enzyme was not inactivated by 5,5'-dithiobis(2-nitrubenzoate), N-e thylmaleimide or iodoacetate, which would point to the absence of an essent ial reactive cysteine residue at the active site. Pyridoxal 5'-phosphate re versibly inactivated the enzyme at pH 7.7 and 5 degrees C, with enzyme acti vity declining to an equilibrium value within 15 min. The remaining activit y depended on the modifier concentration up to about 2 mM. The kinetic anal ysis of inactivation and reactivation fate data is consistent with a revers ible two-step inactivation mechanism with formation of a noncovalent enzyme -pyridoxal 5'-phosphate complex prior to Schiff base formation with a proba ble 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 probab ly to be involved in catalysis.