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Updated: Aug 13, 2026
Enzymatic Cascade Reactions for the Synthesis of Chiral Amino Alcohols from L-lysine
Published on: February 16, 2018
Oxalyl hydroxamates as reaction-intermediate analogues for ketol-acid reductoisomerase
1Central Research and Development Department, E. I. du Pont de Nemours and Company, Wilmington, Delaware 19880-0328.
Abstract:
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
N-Hydroxy-N-isopropyloxamate (IpOHA) strongly inhibits Escherichia coli ketol-acid reductoisomerase, especially with Mg2+ and NADPH. This potent enzyme inhibitor forms a nearly irreversible complex, crucial for understanding bacterial metabolic pathways.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Molecular biology
Background:
- Escherichia coli ketol-acid reductoisomerase is a key enzyme in the branched-chain amino acid biosynthesis pathway.
- Inhibitors of this enzyme can serve as potential antimicrobial agents.
- N-Hydroxy-N-isopropyloxamate (IpOHA) is a known inhibitor, but its precise interaction mechanism requires detailed study.
Purpose of the Study:
- To elucidate the detailed mechanism of inhibition of Escherichia coli ketol-acid reductoisomerase by N-Hydroxy-N-isopropyloxamate (IpOHA).
- To characterize the binding kinetics and affinity of IpOHA to the enzyme under various conditions.
- To investigate the role of divalent metal ions (Mg2+, Mn2+) and nucleotides (NADPH, NADP) in the enzyme-inhibitor interaction.
Main Methods:
- Time-dependent inhibition assays to determine inactivation kinetics.
- Enzyme assays in the presence of varying concentrations of IpOHA, Mg2+, Mn2+, NADPH, and NADP.
- Measurement of association and dissociation rates to calculate inhibition constants and dissociation constants.
Main Results:
- IpOHA exhibits time-dependent, nearly irreversible inhibition of ketol-acid reductoisomerase in the presence of Mg2+ or Mn2+.
- Nucleotides, particularly NADPH, significantly enhance IpOHA binding, forming a tight complex with dissociation constants in the picomolar range (22 pM with Mg2+, 38 pM with Mn2+).
- The enzyme-IpOHA complex stability is highly dependent on the presence and type of nucleotide and divalent metal ion.
Conclusions:
- IpOHA is an exceptionally potent inhibitor of E. coli ketol-acid reductoisomerase, forming a very stable complex with the enzyme.
- The binding mechanism involves a two-step process, and the stability of the inhibited enzyme complex is modulated by cofactors.
- These findings provide critical insights into the enzyme's catalytic mechanism and the development of targeted inhibitors.
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