Aspartate Transcarbamylase: A STUDY OF POSSIBLE ROLES FOR THE SULFHYDRYL GROUP AT THE ACTIVE SITE

Abstract

Abstract Potential roles for the single sulfhydryl group of the catalytic subunit of aspartate transcarbamylase from Escherichia coli have been investigated by means of chemical modification. The S-cyano and S-methyl derivatives are highly active and recombine normally with the zinc regulatory subunit. The derivatives of native enzyme formed in this way are fully responsive to homotropic and heterotropic allosteric effectors and have pH-activity curves indistinguishable from that of unmodified native enzyme. Therefore, despite strong evidence that the—SH group is at the active site, its integrity is not necessary for activity or for allosteric function. However, modifications which lead to negatively charged derivatives of the—SH group do result in inactivation. The reason for such loss of activity was investigated with a sulfonate derivative prepared by rapid and highly specific oxidation of the native enzyme with KMnO4. Carbamyl phosphate and the transition state analog N-(phosphonacetyl)-l-aspartate bind well to the sulfonate derivative, but the affinity of this derivative for succinate (in the presence of carbamyl-P) is much reduced when compared to that of native enzyme. The newly introduced negative charge apparently inactivates by greatly reducing the affinity of the active site for the substrate l-aspartate. The trimeric catalytic subunit is inactive after modification of the 3 identical —SH groups by dithionitrobenzoate, and the derivative is capable of binding tightly only 1 eq of either carbamyl-P or N-(phosphonacetyl)-l-aspartate per trimer, demonstrating a strong interaction among the three active sites of the isolated subunit. Furthermore, the thionitrobenzoate derivative of native aspartate transcarbamylase correspondingly binds only about 2 molecules of N-(phosphonacetyl)-l-aspartate per six active sites and binds them with strong positive cooperativity, suggesting that in unmodified native enzyme positive cooperativity is due at least in part to an interaction between the two catalytic trimers. If catalytic subunit is combined with a limiting amount of zinc regulatory subunit, a new species deficient in regulatory subunit is formed, with altered allosteric properties.