On the mechanism of assembly of the aspartate transcarbamoylase from Escherichia coli.
The mechanism of subunit assembly of aspartate transcarbamoylase from Escherichia coli was studied by following the kinetics of reassociation. The isolated trimetric catalytic subunit (c3) and dimeric regulatory subunit (r2) were mixed together and formation of the dodecameric native enzyme (c6r6) was monitored by measuring changes in activity. Under appropriate conditions the reassociation was second order with respect to the c3 concentration and the effects of varying r2 concentration on the second-order rate constant were examined. An optimum R2 concentration of about 0.07 micrometer was observed. A scheme of the assembly pathways is proposed and is based on the reversible formation of c3r2n (n = 0, 1, 2 or 3) as intermediates. Various combinations of two such c3r2n species are considered as possible rate-limiting steps. This model yields an expression which relates the experimentally determined (overall) second-order rate constant to the equilibrium constant (Kd) governing the formation of c3r2n, the r2 concentration, and four coefficients which reflect the contribution of different types of assembly processes. Using previously determined values of Kd, the above expression for each r2 concentration reduces to a linear equation with four unknowns. The experimental data were subjected to multiple linear-regression analysis and values for the four coefficients were found which gave an excellent fit. Our results show that reassociation of the subunits is a fast bimolecular reaction with rate constants in excess of 10(6) M-1 s-1. Our analysis also suggests that interactions involving a total of more than three r2 subunits (e.g. the combination of c3r2 with c3r6) might contribute significantly to the overall assembly. The influence of various ligands on the reassociation rate profile was also studied. All ligands examined were partially inhibitory to the formation of native enzyme. The effects of substrates were similar to those of CTP whereas the effects of ATP were substantially different. These observations can be readily interpreted by postulating different conformational changes induced by the ligands. These changes should alter the relative orientation of the subunit contacts which must be formed in the reassociation process. The interpretation is consistent with our previous model of the allosteric mechanism.
D001221:Aspartate Carbamoyltransferase / Q000096:biosynthesis*; D001224:Aspartic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D011489:Protein Denaturation; D013386:Succinates / Q000494:pharmacology
D046911:Macromolecular Substances; D013386:Succinates; D001224:Aspartic Acid; D001221:Aspartate Carbamoyltransferase