title: Catalytic-site mapping of pyruvate formate lyase. Hypophosphite reaction on the acetyl-enzyme intermediate affords carbon-phosphorus bond synthesis (1-hydroxyethylphosphonate).
abstract: Pyruvate formate-lyase of Escherichia coli cells, a homodimeric protein of 2 x 85 kDa, is distinguished by the property of containing a stable organic free radical (g = 2.0037) in its resting state. The enzyme (E-SH) achieves pyruvate conversion to acetyl-CoA via two distinct half-reactions (E-SH + pyruvate in equilibrium E-S-acetyl + formate; E-S-acetyl + CoA in equilibrium E-SH + acetyl-CoA), the first of which has been proposed to involve reversible homolytic carbon-carbon bond cleavage [J. Knappe et al. (1984) Proc. Natl Acad. Sci. USA 81, 1332-1335]. Present studies identified Cys-419 as the covalent-catalytic cysteinyl residue via CNBr fragmentation of E-S-[14C]acetyl and radio-sequencing of the isolated peptide CB-Ac (amino acid residues 406-423). Reaction of the formate analogue hypophosphite with E-S-acetyl was investigated and found to produce 1-hydroxyethylphosphonate with a thioester linkage to the adjacent Cys-418. The structure was determined from the chymotryptic peptide CH-P (amino acid residues 415-425), using 31P-NMR spectroscopy (delta = 44 ppm) and by chemical characterisation through degradation into 1-hydroxyethylphosphonate with phosphodiesterase or bromine. This novel P-C-bond synthesis involves the enzyme-based free radical and is proposed to resemble the physiological C-C-bond synthesis (pyruvate production) from formate and E-S-acetyl. These findings are interpreted as proof of a radical mechanism for the action of pyruvate formate-lyase. The central Cys-418/Cys-419 pair of the active site shows a distinctive thiolate property even in the inactive (nonradical) form of the enzyme, as determined using an iodoacetate probe.
authors.affiliation: Institut für Biologische Chemie, Universität Heidelberg, Federal Republic of Germany.
authors.forename: W
authors.initials: W
authors.lastname: Plaga
authors.forename: R
authors.initials: R
authors.lastname: Frank
authors.forename: J
authors.initials: J
authors.lastname: Knappe
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000107:Acetylation; D000123:Acetyltransferases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D003545:Cysteine / Q000378:metabolism; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology; D005561:Formates / Q000378:metabolism; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011773:Pyruvates / Q000378:metabolism
chemical_list: D005561:Formates; D011773:Pyruvates; C030544:formic acid; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; C022560:formate C-acetyltransferase; D003545:Cysteine