RESUMO

Saccharomyces cerevisiae phospho enol pyruvate carboxykinase (EC 4.1.1.49), inactivated by N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine, incorporated 0.95 mol of the fluorescent moiety per mol of enzyme subunit. Reagent incorporation was completely protected by the presence of ADP plus MnCl2. The labeled protein was digested with trypsin after carboxymethylation. Two labeled peptides were isolated by reverse-phase high-performance liquid chromatography and were sequenced by gas-phase automatic Edman degradation. Both peptides contained overlapping amino acid sequences from Asn-358 to Lys-375, thus identifying Cys-364 as the reactive amino acid residue. The position of the target amino acid residue is immediately preceding a putative phosphoryl-binding sequence proposed for some nucleotide-binding proteins.

Assuntos

Cisteína , Fosfoenolpiruvato Carboxiquinase (GTP)/química , Saccharomyces cerevisiae/enzimologia , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Escherichia coli/enzimologia , Corantes Fluorescentes , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Naftalenossulfonatos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Ratos , Homologia de Sequência do Ácido Nucleico , Espectrometria de Fluorescência , Trypanosoma brucei brucei/enzimologia