The location and local environment of the active sites of aspartate transcarbamylase.

The single sulfhydryl of each of the six catalytic chains of aspartate transcarbamylase from Escherichia coli (EC 2.1.3.2) has been modified by reaction with 2chloromercuri-4-nitrophenol (MNP); this treatment results in loss of enzymatic activity. Small thiols can readily remove the bound mercurial. The rate of release of MNP from the catalytic chain sulfhydryls, which are known from x-ray diffraction studies to be within an aqueous central cavity in the molecular complex and from biochemical studies to be near the active sites, is shown to be related to the size of the attacking thiol. For /3-mercaptoethanol and four cysteine analogues, the immobilized mercurial is released from 2 to 4 times more rapidly from isolated catalytic subunit (C,) than from native enzyme (CsRs). The comparatively rigid and bulky B12HllSH-’ (mercaptoundecahydrododecaborate) anion removes MNP 20 times more rapidly from C8 as compared with CsR+ Intermediate results are found for the species CsR4 which is deficient in regulatory subunity (Rz). Steric effects on the rate of displacement of MNP can be accounted for on the basis of partially restricted diffusion of the attacking thiols (and, by extension, substrates and products) into and out of the central cavity, or of conformational differences in the active site regions of the native protein and the free catalytic trimer, or a combination of these phenomena. The rate of /3-mercaptoethanol-induced release of MNP from the catalytic chain sulfhydryl of aspartate transcarbamylase is decreased by a factor of about 2 in the presence of saturating levels of the allosteric inhibitor, cytidine triphosphate (CTP). The activator adenosine triphosphate has little or no effect on the rate of release of MNP, but does antagonize the effect of CTP.