Cysteinyl peptides of rabbit muscle pyruvate kinase labeled by the affinity label 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate

Abstract

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.

Cysteinyl peptides labeled by dibromobutanedione in reaction with rabbit muscle pyruvate kinase

“…The results of this study using dibromobutanedione are in accord with and strengthen our previous conclusions based on the reactions of bromodioxobutyl-nucleotides with pyruvate kinase (Vollmer & Colman, 1990). Cys' @' is a nonessential amino acid in the vicinity of the active site, whereas modification of CysI5' causes loss of activity, suggesting that it is at or near the active site of muscle pyruvate kinase.…”

“…In the case of DBBD, PEP protects best against the inactivation of pyruvate kinase, indicating that reaction occurs in the region of the active site, as it does with the nucleotidyl affinity reagents bearing the bromodioxobutyl arm. The specificity for the active site of the enzyme is likely due to the similarity in structure between the substrate PEP and the dioxobutyl group, which exists predominantly in the enol form (Decamp & Colman, 1989;Vollmer & Colman, 1990). Given that the bromodioxobutyl nucleotides react at the same functional site (that protected by PEP), it is not surprising that the particular residue that reacts with dibromobutanedione causing loss of activity, CYS"~, is the same residue as that which causes loss of activity upon reaction of pyruvate kinase with 8-BDB-TATP (Vollmer & Colman, 1990).…”

“…Single crystal diffractometry at 2.6 A resolution of the cat muscle pyruvate kinase has suggested the location of the active site and of amino acid residues that participate in substrate binding (Muirhead et al, 1986), and this information has been complemented by affinity labeling studies performed using adenine nucleotide analogues that have a chemically reactive group at various positions on the adenine ring (Decamp et al, 1988; D e c a m p & Colman, 1989; Vollmer & Colman, 1990).…”

“…In studies of the reaction of rabbit muscle pyruvate kinase with the nucleotidyl affinity labels 2-BDB-TEA-5'-D P a n d 8-BDB-TA-5'-TP, loss of activity was correlated with reaction at certain residues: Cys'@ and Tyr'47 in the case of ~-B D B -T E A -~' -D P ( D e c a m p & Colman, 1989) and predominantly and CYS'~' in the case of 8-BDB-TA-5'-TP (Vollmer & Colman, 1990). (These residues of the rabbit muscle pyruvate kinase can all be located in the amino acid sequence of the cat muscle enzyme [Muirhead et al, 19861.)…”

Pyruvate Kinase Is Protected by Glutathione-Dependent Redox Balance in Human Red Blood Cells Exposed to Reactive Oxygen Species

“…7). Although it has been indicated that inactivation of rabbit muscle PK was occurred primarily due to modification of cysteine residues in the active site, 30,31) there are little evidences of human PK. Thus, further work is required to elucidate the specific mechanism of inactivation induced by BHP-derived radicals in terms of the modification of active sites or the kinetics changes in order to detail our findings with the data observed in human RBCs under similar conditions.…”