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J. Biol. Chem., Vol. 261, Issue 10, 4499-4503, Apr, 1986

Identification of tyrosine and lysine peptides labeled by 5'-p- fluorosulfonylbenzoyl adenosine in the active site of pyruvate kinase

DL DeCamp and RF Colman

The nucleotide affinity label 5'-p-fluorosulfonylbenzoyl adenosine reacts at the active site of rabbit muscle pyruvate kinase, with irreversible inactivation occurring concomitant with incorporation of about 1 mol of reagent/mol of enzyme subunit (Annamalai, A. E., and Colman, R. F. (1981) J. Biol. Chem. 256, 10276-10283). Purified peptides have now been isolated from 70% inactivated enzyme containing 0.7 mol of reagent/mol of enzyme subunit. Rabbit muscle enzyme labeled with radioactive 5'-p-fluorosulfonylbenzoyl adenosine was digested with thermolysin. Nucleosidyl peptides were purified by chromatography on phenylboronate-agarose and reverse-phase high performance liquid chromatography. After amino acid and N-terminal analysis, the peptides were identified by comparison with the primary sequences of chicken and cat muscle enzyme. About 75% of the reagent incorporated was distributed equally among three O-(4-carboxybenzenesulfonyl)tyrosine- containing peptides: Leu-Asp-CBS-Tyr-Lys-Asn, Val-CBS-Tyr, and Leu-Asp- Asn-Ala-CBS-Tyr. These tyrosines are located in a 28-residue segment of the 530-amino acid sequence. The remainder of the incorporation was found in two N epsilon-(4-carboxybenzenesulfonyl)lysine-containing peptides. Leu-CBS-Lys and Ala-CBS-Lys-Gly-Asp-Tyr-Pro. Modification in the presence of MnATP or MnADP resulted in a marked decrease in labeling of these peptides in proportion to the decreased inactivation. It is suggested that these modified residues are located in the region of the catalytically functional nucleotide binding site of pyruvate kinase.
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