Cysteine 288: an essential hyperreactive thiol of cytosolic phosphoenolpyruvate carboxykinase (GTP)

Phosphoenolpyruvate carboxykinase from the cytosol of rat liver has 13 cysteines, at least one of which is known to be very reactive and essential for catalytic activity (Carlson, G. M., Colombo, G., and Lardy, H. A. (1978) Biochemistry 17, 5329-5338). In order to identify the essential cysteine, this enzyme was modified with the fluorescent sulfhydryl reagent N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide. Incubation of phosphoenolpyruvate carboxykinase with a 10% molar excess of this maleimide at 0 degrees C results in the rapid and nearly complete loss of catalytic activity. Under these conditions, 1 mol of the maleimide is incorporated per mol inactivated enzyme. The substrate GDP provides almost complete protection against inactivation and modification, while phosphoenolpyruvate protects against the rate, but not the extent, of modification. The pH dependence of the rate of enzyme inactivation suggests that the modified residue has a pK alpha of approximately 7.0. Purification and sequencing of the labeled peptide identifies the hyperreactive essential cysteine as Cys-288. This cysteine lies between two putative phosphoryl-binding domains and within a hydrophobic sequence.