Substrate-induced changes in reactivity of sulfhydryl groups of carbamyl phosphate synthetase.

Abstract

1.The influence of various sulfhydryl-group reagents on the activity of frog-liver carbamyl phosphate synthetase (ATP:carbamate phosphotransferase(dephos-phorylating), EC 2.7.2.5) has been examined.2.Acetylglutamate, a cofactor for the enzyme, will accelerate inactivation by some reagents (the disulfide, 5,5′-dithio-bis(2-nitrobenzoic acid); N-ethylmaleimide), but will protect against the action of other reagents (mercuribenzoate; silver). Inactivation by some reagents is only slightly affected by acetylglutamate.3.ATP and Mg2+ will prevent acetylglutamate-induced inactivation by the disulfide, but will not reverse cofactor protection against mercuribenzoate. In the absence of acetylglutamate, ATP and Mg2+ have no effect on inactivation by sulfhydryl reagents.4.Urea and 0·5 M KCl increase the rate of reaction of 5,5′-ditliio-bis(2-nitro-benzoate) with the sulfhydryl groups of the enzyme. Acetylglutamate will do so only in the presence of 0·5 M KCl, or if very low levels of disulfide are used. ATP and Mg21 greatly reduce the rate of reaction of sulfhydryl groups, whether or not cofactor is present. The kinetics are first order in disulfide concentration.5.The present findings strengthen previous findings which suggest that the tertiary protein structure, about the active site of the enzyme, may be modified by cofactor or substrate. It is proposed that mercuribenzoate is highly reactive to one or more sulfhydryl groups which are exposed on the enzyme, but masked by acetylglutamate activation. The cofactor activation, however, exposes other sulfhydryl groups which are preferentially reactive to reagents such as disulfides. Addition of substrate will mask the sulfhydryl groups exposed by activation. The addition of co-factor will fail to influence inactivation by some reagents, because they are equally reactive with both types of sulfhydryl groups. The influence of various sulfhydryl-group reagents on the activity of frog-liver carbamyl phosphate synthetase (ATP:carbamate phosphotransferase(dephos-phorylating), EC 2.7.2.5) has been examined. Acetylglutamate, a cofactor for the enzyme, will accelerate inactivation by some reagents (the disulfide, 5,5′-dithio-bis(2-nitrobenzoic acid); N-ethylmaleimide), but will protect against the action of other reagents (mercuribenzoate; silver). Inactivation by some reagents is only slightly affected by acetylglutamate. ATP and Mg2+ will prevent acetylglutamate-induced inactivation by the disulfide, but will not reverse cofactor protection against mercuribenzoate. In the absence of acetylglutamate, ATP and Mg2+ have no effect on inactivation by sulfhydryl reagents. Urea and 0·5 M KCl increase the rate of reaction of 5,5′-ditliio-bis(2-nitro-benzoate) with the sulfhydryl groups of the enzyme. Acetylglutamate will do so only in the presence of 0·5 M KCl, or if very low levels of disulfide are used. ATP and Mg21 greatly reduce the rate of reaction of sulfhydryl groups, whether or not cofactor is present. The kinetics are first order in disulfide concentration. The present findings strengthen previous findings which suggest that the tertiary protein structure, about the active site of the enzyme, may be modified by cofactor or substrate. It is proposed that mercuribenzoate is highly reactive to one or more sulfhydryl groups which are exposed on the enzyme, but masked by acetylglutamate activation. The cofactor activation, however, exposes other sulfhydryl groups which are preferentially reactive to reagents such as disulfides. Addition of substrate will mask the sulfhydryl groups exposed by activation. The addition of co-factor will fail to influence inactivation by some reagents, because they are equally reactive with both types of sulfhydryl groups.