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

• Published in: Biochemistry (Easton) Vol. 29; no. 11; pp. 2824 - 2830
• Main Authors: Aulabaugh, Ann, Schloss, John V
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 20.03.1990
• Subjects: 550201 - Biochemistry- Tracer Techniques; Alcohol Oxidoreductases - antagonists & inhibitors; Alcohol Oxidoreductases - isolation & purification; Alcohol Oxidoreductases - metabolism; AMINES; Analytical, structural and metabolic biochemistry; BASIC BIOLOGICAL SCIENCES; BIOCHEMICAL REACTION KINETICS; Biological and medical sciences; CARBON 14 COMPOUNDS; CARBOXYLIC ACIDS; Chemical Phenomena; Chemistry; DICARBOXYLIC ACIDS; Enzyme Activation - drug effects; ENZYME INHIBITORS; ENZYMES; Enzymes and enzyme inhibitors; Escherichia coli - drug effects; Escherichia coli - enzymology; Fundamental and applied biological sciences. Psychology; HEMIACETAL DEHYDROGENASES; HYDROXAMIC ACIDS; Hydroxamic Acids - pharmacokinetics; HYDROXY COMPOUNDS; ISOMERASES; Ketol-Acid Reductoisomerase; KINETICS; LABELLED COMPOUNDS; Magnesium - metabolism; Molecular Structure; N-hydroxy-N-isopropyloxamate; NAD - metabolism; ORGANIC ACIDS; ORGANIC COMPOUNDS; Oxalates - pharmacokinetics; OXALIC ACID; OXIDOREDUCTASES; REACTION INTERMEDIATES; REACTION KINETICS; Reaction intermediate; Enzyme; Escherichia coli; Association constant; Enzyme inhibitor; Metal Ions; Dissociation constant; NMR spectrometry; Ion exchange chromatography; Transition state; Reaction mechanism; Nucleotide; Bacteria; Ketol-acid reductoisomerase; Kinetic parameter; Enterobacteriaceae
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00463a027

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 beta-keto acid into an alpha-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 10(4) M-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 10(4) M-1 s-1 and 3.2 X 10(-6) 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.