ACCELERATOR MASS-SPECTROMETRY FOR ASSAYING IRREVERSIBLE COVALENT MODIFICATION OF AN ENZYME BY ACETOACETIC ESTER

Protein modification (sometimes known as crosslinking) often requires two or more steps to affix a small molecule irreversibly. Two-step red uctive alkylation of the enzyme rabbit muscle aldolase with ethyl 3-C- 14-acetoacetate and sodium cyanoborohydride attaches less radioactivit y than with cyanoborohydride omitted. The C-14 level incorporated into aldolase corresponds to only about 15-30 modified protein molecules p er million. Accelerator mass spectrometry (AMS) provides the only tech nique currently available for investigating the shorter chains from CN Br-cleavage of modified aldolase. Examination of individual fragments reveals that reductive alkylation of the active site lysine in the pre sence of cyanoborohydride (+BH3CN) is negligible when compared with th e extent of covalent modification in the absence of cyanoborohydride ( -BH3CN). Labeling by ethyl acetoacetate cannot result from simple acet oacetylation, because dialysis with hydroxylamine does not wash it out . The amount of C-14 incorporated from ethyl 3-C-14-acetoacetate witho ut cyanoborohydride is roughly proportional to the number of tyrosine residues in each CNBr-fragment, and we surmise that ethyl acetoacetate attaches irreversibly via a reaction specific to that amino acid. Cya noborohydride inhibits this reaction, but appears to diminish the susc eptibility of the active site tyrosine (which is close to a lysine in the tertiary structure of aldolase) less than other tyrosine residues. (Int J Mass Spectrom 179/180 (1998) 185-193) (C) 1998 Elsevier Scienc e B.V.