OXALYL HYDROXAMATES AS REACTION-INTERMEDIATE ANALOGS FOR KETOL-ACID REDUCTOISOMERASE

N-Hydroxy-.N-isopropyloxamate (IpOHA) is an exceptionally potent inhibitor of the Escherichia coli ketol-acid reductoisomerase. In the presence of Mg2+ or Mn2+, IpOHA inhibits the enzyme in a time-dependent manner, forming a nearly irreversible complex. Nucleotide, which is essential for catalysis, greatly enhances the binding of IpOHA by the reductoisomerase, with NADPH (normally present during the enzyme's rearrangement step, i.e., conversion of a β-keto acid into an α-keto acid, in either the forward or reverse physiological reactions) being more effective than NADP. In the presence of Mg2+ and NADPH, IpOHA appears to bind to the enzyme in a two-step mechanism, with an initial inhibition constant of 160 nM and a maximum rate of formation of the tight, slowly reversible complex of 0.57 min−1 (values that give an association rate of IpOHA, at low concentration, of 5.9 x 104 NT−1 s−1)- The rate of exchange of [14C]IpOHA from an enzyme-[14C]IpOHA-Mg2+-NADPH complex with exogenous, unlabeled IpOHA has a half-time of 6 days (150 h). This dissociation rate (1.3 X 10−6 s−1) and the association rate determined by inactivation kinetics define an overall dissociation constant of 22 pM. By contrast, in the presence of Mn2+ and NADPH, the corresponding association and dissociation rates for IpOHA are 8.2 X 104 M−1 s−1 and 3.2 x 106 s−1 (half-time = 2.5 days), respectively, which define an overall dissociation constant of 38 pM. In the presence of NADP or in the absence of nucleotide (both in the presence of Mg2+), the enzyme-IpOHA complex is far more labile, with dissociation half-times of 28 and 2 h, respectively. In the absence of Mg2+ or Mn2+, IpOHA does not exhibit time-dependent inhibition of the reductoisomerase. These results parallel the effects that divalent metals and nucleotide have on the rearrangement step of this enzyme, which is greater than 3-fold more rapid in the presence of NADPH than in the presence of NADP and absolutely dependent on Mg2+, and strongly suggest that IpOHA is a potent inhibitor of ketol-acid reductoisomerase by virtue of its structural similarity to the rearrangement transition state. © 1990, American Chemical Society. All rights reserved.