MECHANISM FOR THE INHIBITION OF ALDEHYDE DEHYDROGENASE BY NITRIC-OXIDE

The inhibition of Saccharomyces cerevisiae aldehyde dehydrogenase (AlD H) by gaseous nitric oxide (NO) in solution and by NO generated from d iethylamine nonoate was time and concentration dependent. The presence of oxygen significantly reduced the extent of inhibition by NO, indic ating that NO itself rather than an oxidation product of NO such as N2 O3 is the inhibitory species under physiological conditions. A cystein e residue at the active site of the enzyme was implicated in this inhi bition based on the following observations: a) NAD(+) and NADP(+), but not reduced cofactors, significantly enhanced inhibition of AlDH by N O; b) the aldehyde substrate, benzaldehyde, blocked inhibition; and c) inhibition was accompanied by loss of free sulfhydryl groups on the e nzyme. Activity of the NO-inactivated enzyme was readily restored by t reatment with dithiothreitol (DTT), but not with GSH. This difference was attributed, in part. to a redox process leading to the formation o f a cyclic DTT disulfide. Based on the chemistry deduced from model sy stems, the reaction of NO with AlDH sulfhydryls was shown to produce i ntramolecular disulfides and N2O. These disulfides were shown to be in trasubunit disulfides by nonreducing SDS-PAGE analysis of the NO-inhib ited enzyme. Following complete inhibition of AlDH by NO, four of the eight titratable (Ellman's reagent) sulkydryl groups of AlDH were foun d to be oxidized to disulfides. These results suggest that a) the sulf hydryl group of active site Cys-302 and a proximal cysteine are oxidiz ed to form an intrasubunit disulfide by NO; b) only two of the four su bunits of ALDH art: catalytically active; and c) NO preferentially oxi dizes sulfhydryl groups of the catalytically active subunits. A detail ed mechanism for the inhibition of AlDH by NO is presented. (C) 1997 E lsevier Science Inc.