REACTIVITY OF CYSTEINYL, ARGINYL, AND LYSYL RESIDUES OF ESCHERICHIA-COLI PHOSPHOENOLPYRUVATE CARBOXYKINASE AGAINST GROUP-SPECIFIC CHEMICAL REAGENTS

Calcium-activated phosphoenolpyruvate carboxykinase from Escheria coil is not inactivated by a number of sulfhydryl-directed reagents [5,5'- dithiobis(2-nitrobenzoate), iodoacetate, N-ethylmaleimide, N-(1-pyreny l)maleimide or oacetyl)-N'-(5-sulfo-1-naphthylethylene-diamine)], unli ke phosphoenolpyruvate carboxykinase from other organisms. On the othe r hand, the enzyme is rapidly inactivated by the arginyl-directed reag ents 2,3-butanedione and 1-pyrenylglyoxal. The substrates, ADP plus PE P in the presence of Mn2+, protect the enzyme against inactivation by the diones. Quantitation of pyrenylglyoxal incorporation indicates tha t complete inactivation correlates with the binding of one inactivator molecule per mole of enzyme. Chemical modification by pyridoxal 5'-ph osphate also produces inactivation of the enzyme, and the labeled prot ein 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 a nd 0.30 mol of reagent incorporated per mole of enzyme were found in t he absence and presence of substrates, respectively. The results sugge st the presence of functional arginyl and lysyl residues in or near th e active site of the enzyme, and indicate lack of reactive functional sulfhydryl groups.