Catalytic‐site mapping of pyruvate formate lyase

Abstract

Pyruvate formate‐lyase of Escherichia coli cells, a homodimeric protein of 2 × 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 ⇌ E‐S‐acetyl + formate; E‐S‐acetyl + CoA ⇌ 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‐ [ 14 C]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‐hydroxyethyl‐phosphonate 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 31 P‐NMR spectroscopy (δ= 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.