Identification of a Reactive Cysteine Residue at the Glutamine Binding Site of Carbamyl Phosphate Synthetase

Abstract

Abstract Carbamyl phosphate synthetase from Escherichia coli, which is composed of a light and a heavy subunit, is inhibited with respect to its glutamine-dependent reactions by treatment with l-2-amino-4-oxo-5-chloro[5-14C]pentanoic acid. This glutamine analog reacts with a glutamine- or albizziinprotectable site on the light subunit to yield an enzyme covalently linked to a 4-oxo[14C]norvaline moiety. The molar ratio of bound 14C to the light subunit is close to unity; some binding also occurs on the heavy subunit, but this is noninhibitory. The chloroketone-treated enzyme exhibits increased ATPase activity and a decreased apparent Km value for NH4Cl. Mg++ATP protects the enzyme significantly against chloroketone; allosteric effectors (ornithine, IMP, UMP) offer little or no protection. After reaction of the enzyme with the chloroketone, the enzyme was treated with performic acid and then subjected to acid hydrolysis; S-[14C]carboxymethylcysteine was isolated from such hydrolysates of the labeled enzyme and the isolated labeled light subunit. This evidence indicates that the chloroketone reacts with a sulfhydryl group at the glutamine site of the light subunit. This sulfhydryl group does not react with N-ethylmaleimide at neutral pH which suggests that it is buried. The protective effect of Mg++ATP against chloroketone, the increase in ATPase activity on treatment with chloroketone, and the decreased apparent Km for NH4Cl may reflect functional intersubunit interactions associated with the binding of glutamine and ATP to the light and heavy subunits, respectively, in the native enzyme.