Reactivity of cysteinyl, arginyl, and lysyl residues ofEscherichia coli phosphoenolpyruvate carboxykinase against group-specific chemical reagents

Abstract

Calcium-activated phosphoenolpyruvate carboxykinase from Escherichia coli is not inactivated by a number of sulfhydryl-directed reagents [5,5'-dithiobis(2-nitrobenzoate), iodoacetate, N-ethylmaleimide, N-(1-pyrenyl)maleimide or N-(iodoacetyl)-N'-(5-sulfo-1-naphthylethylenediamine)], unlike phosphoenolpyruvate carboxykinase from other organisms. On the other hand, the enzyme is rapidly inactivated by the arginyl-directed reagents 2,3-butanedione and 1-pyrenylglyoxal. The substrates, ADP plus PEP in the presence of Mn2+, protect the enzyme against inactivation by the diones. Quantitation of pyrenylglyoxal incorporation indicates that complete inactivation correlates with the binding of one inactivator molecule per mole of enzyme. Chemical modification by pyridoxal 5'-phosphate also produces inactivation of the enzyme, and the labeled protein shows a difference spectrum with a peak at 325 nm, characteristic of a pyridoxyl derivative of lysine. The inactivation by this reagent is also prevented by the substrates. Binding stoichiometries of 1.25 and 0.30 mol of reagent incorporated per mole of enzyme were found in the absence and presence of substrates, respectively. The results suggest the presence of functional arginyl and lysyl residues in or near the active site of the enzyme, and indicate lack of reactive functional sulfhydryl groups.

Woodward's reagent K reacts with histidine and cysteine residues inEscherichia coli andSaccharomyces cerevisiae phosphoenolpyruvate carboxykinases

“…In this work, we have used as models Escherichia coli and Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases, two enzymes of current use in our laboratory, whose structure and chemical modification characteristics are well known (Matte et al, 1996;Bazaes et al, 1993;Krautwurst et al, 1996). Our findings confirm the work of Llamas et al (1986) and indicate that WRK modifies histidyl and cysteinyl groups in these enzymes.…”

“…This location agrees with the observed protection of inactivation and labeling afforded by ADP+PEP+Mn 2+. The lack of reaction of WRK with cysteinyl residues in this enzyme is in line with the known low reactivity of the enzyme sulfhydryls against a range of group-specific reagents (Bazaes et al, 1993).…”

“…The rate of oxaloacetate formation was determined spectrophotometrically at 30~ as described by Malebrfin and Cardemil (1987) for the yeast enzyme and Bazaes et al (1993) for the bacterial PEPCK.…”

Molecular and expression analysis of the Rhizobium meliloti phosphoenolpyruvate carboxykinase (pckA) gene

“…Biochemical analysis of the yeast Pck enzyme has identified two reactive cysteines (Cys-364 and Cys-457), present in the GTP-dependent Pcks, whose chemical alteration resulted in enzyme inactivation (3). These two residues are not conserved among the ATP-dependent Pcks and biochemical analysis of the E. coli Pck did not identify any reactive cysteine (8), which would suggest that these cysteines are not required per se for the enzyme function.…”

Structure and Mechanism of Phosphoenolpyruvate Carboxykinase

“…Despite this lack of overall homology, both groups of PCKs contain similar NTP and oxaloacetate binding "consensus motifs" in their active sites, which likely play similar roles in substrate binding (which will be described in this review). Also, both GTP-dependent and ATP-dependent PCKs have been shown to possess lysinyl (13)(14)(15)(16)(17), argininyl (18,19), and histidinyl (20,21) residues at or near their active sites. The differences in nucleotide specificity and kinetic properties between ATP-and GTP-dependent PCKs have led to the suggestion that they may be potential therapeutic targets in parasitic nematodes (22) and in Trypanomastid parasites such as T. cruzi (11).…”