2-[(4-Bromo-2,3-dioxobutyl)thio]-1, N6-ethenoadenosine 5′-diphosphate: A new fluorescent affinity label of a tyrosyl residue in the active site of rabbit muscle pyruvate kinase

Abstract

A new reactive fluorescent ADP analog has been synthesized: 2-[(4-bromo-2,3-dioxobutyl)thio]-1,N6-ethenoadenosine 5'-diphosphate (2-BDB-T epsilon A-5'-DP). Rabbit muscle pyruvate kinase is inactivated by 200 microM 2-BDB-T epsilon A-5'-DP in a biphasic manner, with an initial loss of 75% activity followed by a slow total inactivation. The rate constants for both phases exhibit nonlinear dependence on reagent concentration, consistent with reversible formation of an enzyme-reagent complex (KI = 133 microM) prior to irreversible reaction. Loss of activity is prevented by substrates. The best protection against inactivation is provided by phosphoenolpyruvate (PEP), KCl, and MnSO4, suggesting that the reaction occurs in the region of the PEP binding site. Incorporation of 1.7 mol/mol enzyme subunit accompanies 90% inactivation by 200 microM 2-BDB-T epsilon A-5'-DP in 80 min. However, in the presence of PEP, KCl, and MnSO4, 1.0 mol of reagent is incorporated when the enzyme is only 14% inactivated. These results indicate that 2-BDB-T epsilon A-5'-DP reacts with two groups on the enzyme, one of which is at or near the PEP binding site. Incubation of pyruvate kinase with related nucleotide analogs lacking a 5'-diphosphate or a diketo group suggests that the diketo group, but not the diphosphate, is essential for inactivation. The enolized form of the bromodioxobutyl group resembles phosphoenolpyruvate and probably directs the reagent to the PEP binding site. Modified enzyme, prepared by incubating pyruvate kinase with 200 microM 2-BDB-T epsilon A-5'-DP in the absence and presence of phosphoenolpyruvate, KCl, and MnSO4, was reduced with [3H]NaBH4, carboxymethylated, and digested with trypsin. Nucleotidyl peptides were isolated by chromatography on phenylboronateagarose followed by reverse phase high pressure liquid chromatography. Two radioactive peptides were identified: Asn162-Ile-Cys-Lys165 and Ile141-Thr-Leu-Asp-Asn-Ala-Tyr-Met-Glu-Lys150. Only the tetrapeptide was modified in the presence of PEP, KCl, and Mn+ when the enzyme retained most of its activity. Cys164 is thus designated the nonessential modified residue, while modification of Tyr147 near the active site of pyruvate kinase is responsible for loss of enzymatic activity. The observed biphasic kinetics of inactivation are due to the negatively cooperative reaction of 2-BDB-T epsilon A-5'-DP with Tyr147 in the tetramer. The new compound, 2-BDB-T epsilon A-5'-DP, may have general application as an affinity label of ADP and PEP sites in other proteins.