The thiol group in the catalytic chains of aspartate transcarbamoylase.
The allosteric enzyme aspartate transcarbamoylase (EC 2.1.3.2) was previously shown to consist of two functionally distinct types of polypeptide chains. X-ray diffraction and chemical studies showed that there are six copies of both catalytic (C) and regulatory (R) chains, and that the intact molecular complex (C(6)R(6)) has D(3) symmetry. Organomercurials react preferentially with the four thiol groups on each R chain, dissociating the molecular complex. We show that 2-chloromercuri-4-nitrophenol reacts specifically and rapidly with the single C-chain thiol, which is believed to be near the catalytic site. This reaction inactivates the enzyme in solution and does not dissociate the molecular complex. Spectrophotometric titration and mercury analysis indicates that six molecules of this mercurial are firmly bound to the enzyme (R(6)C(6)), and crystallographic studies establish that only six sites, related by D(3) symmetry, are modified. The known low reactivity of this C-chain thiol with other sulfhydryl reagents, the unusual structural requirements in the reaction with 2-chloromercuri-4-nitrophenol, and the spectral properties of the resulting derivative provide insight into the environment of this thiol. Probably, at least one positively charged group of the enzyme is nearby, and the environment of this thiol is at least partially hydrophobic.
D000494:Allosteric Regulation; D001221:Aspartate Carbamoyltransferase / Q000032:analysis* / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D002713:Chlorine; D003461:Crystallography; D004926:Escherichia coli / Q000201:enzymology; D006902:Hydroxymercuribenzoates / Q000378:metabolism; D007700:Kinetics; D008628:Mercury / Q000378:metabolism; D009596:Nitrophenols / Q000378:metabolism; D009942:Organometallic Compounds / Q000378:metabolism; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000032:analysis*; D014961:X-Ray Diffraction
D006902:Hydroxymercuribenzoates; D009596:Nitrophenols; D009942:Organometallic Compounds; D013438:Sulfhydryl Compounds; D002713:Chlorine; D001221:Aspartate Carbamoyltransferase; D008628:Mercury