Inactivation of Escherichia coli pyruvate formate-lyase by hypophosphite: evidence for a rate-limiting phosphorus-hydrogen bond cleavage

Journal Article · Tue Mar 22 04:00:00 EST 1988 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00406a061· OSTI ID:7126076

Brush, E J; Lipsett, K A; Kozarich, J W

Recently, Knappe and co-workers have shown that the catalytically active form of pyruvate formate-lyase from Escherichia coli is associated with a protein-bound organic free radical which is quenched upon enzyme inactivation by oxygen or hypophosphite. Our interest in the chemical mechanism of this unusual enzymatic reaction has led us to investigate several key aspects of the inactivation of the lyase by hypophosphite and its relationship to the normal enzymatic reaction. We report here that the inactivation of both the free and acetylated forms of the lyase is subject to a primary kinetic isotope effect using (2H2)hypophosphite. This suggests that phosphorus-hydrogen bond cleavage is at least partially rate limiting during inactivation. In addition, the inactivated enzyme can be fully reactivated. We have also determined a Vmax/Km isotope effect of 3.6 +/- 0.7 for pyruvate formation from (2H)formate and acetyl coenzyme A. Thus, carbon-hydrogen bond cleavage is partially rate limiting in the normal reverse reaction. On the basis of our findings, the previous work of Knappe and co-workers, the likelihood that hypophosphite is a formate analogue, the known susceptibility of both hypophosphite and formate to homolysis, and a chemical precedent for homolytic cleavage of pyruvate, we offer a preliminary mechanistic proposal for the lyase reaction.

• Research Organization: Univ. of Maryland, College Park (USA)
• OSTI ID: 7126076
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 27:6; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL EFFECTS
CHEMICAL BONDS
CLEAVAGE
CRYSTAL STRUCTURE
DEUTERIUM
ELEMENTS
ENZYME REACTIVATION
ENZYMES
ESCHERICHIA COLI
HYDROGEN ISOTOPES
INHIBITION
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
LYASES
MICROORGANISMS
MICROSTRUCTURE
NONMETALS
NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC PHOSPHORUS COMPOUNDS
OXYGEN
PHOSPHINIC ACIDS
RADICALS
REACTION KINETICS
STABLE ISOTOPES
TRACER TECHNIQUES