CYSTEINYL PEPTIDES OF RABBIT MUSCLE PYRUVATE-KINASE LABELED BY THE AFFINITY LABEL 8-[(4-BROMO-2,3-DIOXOBUTYL)THIO]ADENOSINE 5'-TRIPHOSPHATE

The affinity label 8-[(4-bromo-2,3-dioxobutyl)thio] adenosine 5′-triphosphate (8-BDB-TA-5′-TP) reacts covalently with rabbit muscle pyruvate kinase, incorporating 2 mol of reagent/mol of enzyme subunit upon complete inactivation. Protection against inactivation is provided by phosphoenolpyruvate, K+, and Mn2+ and only 1 mol of reagent/mol of subunit is incorporated [DeCamp, D. L., Lim, S., & Colman, R. F. (1988) Biochemistry 27, 7651–7658]. We have now identified the resultant modified residues. After reaction with 8-BDB-TA-5′-TP at pH 7.0, modified enzyme was incubated with [3H]NaBH4 to reduce the carbonyl groups of enzyme-bound 8-BDB-TA-5′-TP and to introduce a radioactive tracer into the modified residues. Following carboxymethylation and digestion with trypsin, the radioactive peptides were separated on a phenylboronate agarose column followed by reverse-phase high-performance liquid chromatography in 0.1% trifluoroacetic acid with an acetonitrile gradient. Gas-phase sequencing gave the cysteine-modified peptides Asn162-Ile-Cys-Lys165 and Cys151-Asp-Glu-Asn-Ile-Leu-Trp-Leu-Asp-Tyr-Lys161, with a smaller amount of Asn43-Thr-Gly-Ile-Ile-Cys-Thr-Ile-Gly-Pro-Ala-Ser-Arg55. Reaction in the presence of the protectants phosphoenolpyruvate, K+, and Mn2+ yielded Asn-Ile-Cys-Lys as the only labeled peptide, indicating that inactivation is caused by modification of Cys151 and Cys48. © 1990, American Chemical Society. All rights reserved.