1. Some kinetic properties of aspartate transcarbamoylase (EC 2.1.3.2), that had been purified approx. 20-fold from wheat germ, were studied. 2. A plot of enzyme activity against pH showed a low maximum at pH8.4 and a second, higher, maximum at pH10.5. A plot of percentage inhibition by 0.2mm-UMP against pH was approximately parallel to the plot of activity against pH, except that between pH6.5 and 7.5 the enzyme was insensitive to 0.2mm-UMP. 3. Kinetics were studied in detail at pH10.0 and 25 degrees C. In the absence of UMP, initial-rate plots were hyperbolic when the concentration of either substrate was varied. UMP decreased both V(max.) and K(m) in plots of initial rate against l-aspartate concentration, but the plots remained hyperbolic. However, UMP converted plots of initial rate against carbamoyl phosphate concentration into a sigmoidal shape, without significantly affecting V(max.). Plots of initial rate against UMP concentration were also sigmoidal. 4. The theoretical model proposed by Monod et al. (1965) gave a partial explanation of these results. When quasi-equilibrium conditions were assumed analysis in terms of this model suggested a trimeric enzyme binding the allosteric ligands, carbamoyl phosphate and UMP, nearly exclusively to the R and T conformational states respectively, and existing predominantly in the R state when ligands were absent. However, the values of the Hill coefficients for the co-operativity of each allosteric ligand were somewhat less than those predicted by the theory. 5. Some of the implications of these results are discussed, and the enzyme is contrasted with the well-known aspartate transcarbamoylase of Escherichia coli.

The kinetic effects of the end-product inhibitor UMP on aspartate transcarbamoylase (EC 2.1.3.2) purified to homogeneity from wheat germ were studied. In agreement with an earlier study of the relatively crude enzyme [Yon (1972) Biochem. J. 128, 311-320], the half-saturating concentrations of UMP and of the first substrate, carbamoyl phosphate (but not of the second, L-aspartate), were found to be strongly interdependent. However, the kinetic behaviour of the pure enzyme differed from that of the crude enzyme in several important respects, namely: (a) the apparent affinity for UMP was lower with the pure enzyme; (b) sigmoidicity was absent from plots of initial rate versus carbamoyl phosphate concentration, each at a fixed UMP concentration; (c) sigmoidicity was greatly exaggerated in plots of initial rate versus UMP concentration, each at a fixed carbamoyl phosphate concentration, owing to the occurrence of a slight but definite maximum in each plot at low UMP concentration; (d) there was a relative increase in this maximum in the presence of N-phosphonacetyl-L-aspartate, an inhibitor competitive with carbamoyl phosphate. It is shown that a modified two-conformation concerted-transition model can be used to account for most of these features of the pure enzyme. The model treats carbamoyl phosphate and UMP as antagonistic allosteric ligands binding to alternative conformational states [Monod, Wyman & Changeux (1965) J. Mol. Biol. 12, 88-118], carbamoyl phosphate binding non-exclusively (dissociation constants 20 microM and 85 microM respectively) and UMP binding exclusively (dissociation constant 2.5 microM). The model postulates further that the conformation with lower affinity for carbamoyl phosphate has the higher value of kcat., and that it binds UMP in competition with carbamoyl phosphate. Parameters giving the best fit of experimental data to this model were found by a non-linear least-squares search procedure.

1. The molecular mass of aspartate transcarbamoylase purified from wheat germ was found to be 101kDa by sucrose-density-gradient centrifugation, 103kDa by gel-filtration chromatography and 108kDa by polyacrylamide-gel electrophoresis. A mean value of 104 +/- 11kDa was obtained by pooling several replicate results from each method. 2. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate indicated a single size of polypeptide chain of mean molecular mass 37 +/- 4kDa. The ratio of the mean molecular masses of the active and denatured enzymes is 2.8.3. When the active enzyme was covalently cross-linked at a low protein concentration by dimethyl suberimidate, and then examined electrophoretically under denaturing conditions, three size species were observed to predominate, of apparent molecular masses 36, 77 and 106kDa respectively. 4. These results indicate that the intact, fully regulatory enzyme is a simple trimer, slightly larger than the trimeric "catalytic subunit' of the aspartate transcarbamoylase from Escherichia coli [Weber (1968) Nature (London) 218, 1116-1118]. The prevalence of trimeric structures amongst carbamoyl-transferase enzymes is discussed.

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1. The steady-state kinetics of the bisubstrate reaction catalysed by aspartate transcarbamoylase purified from wheat (Triticum vulgare)-germ have been studied at 25 degrees C, pH 8.5 AND I 0.10-0.12. Initial-velocity and product-inhibition results are consistent with an ordered sequential mechanism in which carbamoyl phosphate is the first substrate to bind, followed by L-aspartate, and carbamoyl aspartate is the first product to leave, followed by Pi. The order of substrate addition is supported by dead-end inhibition studies using pyrophosphate and maleate as inhibitory analogues of the substrates. Product inhibition permitted a minimum value for the dissociation constant of L-aspartate from the ternary complex to be estimated. This minimum is of the same order as the dissociation constant (Ki) of succinate. 2. A range of dicarboxy analogues of L-aspartate were tested as possible inhibitors of the enzyme. These studies suggested that L-aspartate is bound with its carboxy groups in the eclipsed configuration, and that the stereochemical constraints around the binding site are very similar to those reported for the catalytic subunit of the enzyme from Escherichia coli [Davies, Vanaman & Stark (1970) J. Biol. Chem. 245, 1175-1179].