Identification of cysteine as the reactive group in pyruvate kinase alkylated by 5-chloro-4-oxopentanoic acid

Chalkley, R A; Bloxham, David P.

doi:10.1042/bj1590213

Cited by 7 publications

(1 citation statement)

References 10 publications

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“…9.2. This value is virtually identical with the pKa for a similar covalent reaction involving the nucleophilic displacement of halogen from 5chloro-4-oxopentanoic acid by Cys-3 15 in the muscle pyruvate kinase sequence (Chalkley & Bloxham, 1976;Bloxham et al, 1978; deduced from chicken gene sequence of M-type pyruvate kinase determined by Lonberg & Gilbert, 1983). None of Mg2+ (4mM), ADP (0.5mM), ATP Table 2.…”

Section: Stoicheiometry Of Modification Of L-type Pyruvate Kinasesupporting

confidence: 54%

Specific inhibition of L-type pyruvate kinase by the triazine dye Procion Blue MX-R.

Byford¹,

Bloxham²

1984

Biochemical Journal

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Incubation of the triazine dye Procion Blue MX-R with L- and M-type pyruvate kinase resulted in rapid time- and dye-concentration-dependent loss of activity. L-type pyruvate kinase was protected only by a low concentration of Mg2+; this was not the case with the M-type enzyme. Modification of the L-type form resulted in the incorporation of 1.54 +/- 0.057 mol of dye/mol of enzyme subunit in the absence of Mg2+, but only 0.73 +/- 0.024 mol of dye/mol of enzyme subunit in the presence of Mg2+. Tryptic peptide mapping of L-type pyruvate kinase modified in the presence and in the absence of Mg2+ further indicated that there were two sites modified in the enzyme, one of which was protected by Mg2+. The pKa of the nucleophile involved in the modification was calculated to be 7.1, implicating the possible involvement of a histidine residue. L-type enzyme was bound to Sepharose-immobilized Procion Blue MX-R specifically in the presence of Mg2+, whereas binding of the M-type enzyme was Mg2+-independent. The specific interaction of L-type pyruvate kinase with the dye was exploited in the large-scale purification of the enzyme and in the isolation of the phosphorylated enzyme.

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Section: Stoicheiometry Of Modification Of L-type Pyruvate Kinasesupporting

confidence: 54%