PARACATALYTIC SELF-INACTIVATION OF FRUCTOSE-1,6-BISPHOSPHATE ALDOLASE- STRUCTURE OF THE CROSS-LINK FORMED AT THE ACTIVE-SITE

Oxidation of enzyme-substrate carbanion intermediates by extrinsic oxi dants may result in irreversible paracatalytic inactivation of certain enzymes. In paracatalytically modified fructose-1,6-bisphosphate aldo lase from rabbit muscle the polypeptide chain had been found to be cro sslinked at active-site Lys229 (Schiff base forming with substrate) an d Lys146 by a phosphorylated three-carbon moiety [Lubini, D. G. E. and Christen, P. (1979) Proc. Natl Acad. Sci. USA 76,2527-2531]. In the p resent study, the structure of this crosslink was elucidated by instru mental analysis. Aldolase was paracatalytically modified in the presen ce of fructose 1,6-bisphosphate and hexacyanoferrate(III). The complet ely inactivated enzyme was digested with pronase. The crosslinked pept ide was isolated by gel filtration and reverse-phase HPLC. Mass spectr oscopy, H-1- and C-13-NMR showed that a derivative of dihydroxyacetone phosphate forms an amidine with the epsilon-amino groups of die two l ysine residues: [GRAPHICS]