Oxalyl hydroxamates as reaction-intermediate analogues for ketol-acid reductoisomerase

Journal Article · Mon Mar 19 23:00:00 EST 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00463a027· OSTI ID:6325456

Aulabaugh, A; Schloss, J V ^[1]

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1. E.I. du Pont De Nemours and Co., Wilmington, DE (USA)

N-Hydroxy-N-isopropyloxamate (IpOHA) is an exceptionally potent inhibitor of the Escherichia coli ketol-acid reductiosomerase. In the presence of Mg{sup 2+} or Mn{sup 2+}, 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 being more effective than NADP. In the presence of Mg{sup 2+} and NADPH, IpOHA appears to bind to the enzyme in a two-step mechanism. The rate of exchange of ({sup 14}C)IpOHA from an enzyme-({sup 14}C)IpOHA-Mg{sup 2+}-NADPH complex with exogenous, unlabeled IpOHA has a half-time of 6 days. This dissociation rate and the association rate determined by inactivation kinetics define an overall dissociation constant of 22 pM. By contrast, in the presence of Mn{sup 2+} and NADPH, the corresponding association and dissociation rates for IpOHA are 8.2 {times} 10{sup 4} M{sup {minus}1} s{sup {minus}1} and 3.2 {times} 10{sup {minus}6} s{sup {minus}1}, respectively, which define an overall dissociation constant of 38 pM. In the presence of NADP or in the absence of nucleotide, the enzyme-IpOHA complex is far more labile, with dissociation half-times of 28 and 2 h, respectively. 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 Mg{sup 2+}, and strongly suggest that IpOHA is a potent inhibitor of ketol-acid reductoisomerase by virtue of its structural similarity to the rearrangement transition state.

• OSTI ID: 6325456
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 29:11; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINES
BIOCHEMICAL REACTION KINETICS
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
DICARBOXYLIC ACIDS
ENZYME INHIBITORS
ENZYMES
HEMIACETAL DEHYDROGENASES
HYDROXAMIC ACIDS
HYDROXY COMPOUNDS
ISOMERASES
KINETICS
LABELLED COMPOUNDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
OXALIC ACID
OXIDOREDUCTASES
REACTION INTERMEDIATES
REACTION KINETICS