Is substrate inhibition a consequence of allostery in aspartate transcarbamylase?

Aspartate transcarbamylase (ATCase) is a highly regulated, multisubunit enzyme that catalyzes the first regulated step in pyrimidine biosynthesis. Although ATCase exhibits strong substrate inhibition (the reduction of enzyme activity at high substrate concentrations), the mechanism of substrate inhibition has not been investigated. At the molecular level, substrate inhibition may result either from local events at the active site or from global or specific long-range allosteric effects. We have compared the results of fitting kinetic data to several models: (a) a semi-empirical steady-state kinetic model that includes cooperative substrate binding (described by a Hill coefficient) and partial uncompetitive substrate inhibition, (b) a nested allosteric model developed to analyze substrate inhibition of the ATPase activity of GroEL, an enzyme with a quaternary structure analogous to ATCase (O. Yifrach and A. Horovitz, Biochemistry, 34 (1995) 5303), and (c) purely concerted models, including a model originally proposed by Monod et al. (J. Monod, J. Wyman and J.P. Changeux, J. Mol. Biol., 12 (1965) 88). Model (a) is the first kinetic equation for ATCase that both fits the data and returns physically realistic values for all parameters, but it is a modified Hill equation and thus returns little or no molecular mechanistic information. The nested allosteric model (b), which assumes concerted cooperativity within each catalytic trimer of ATCase and sequential cooperativity between trimers, is unlikely to be the correct model for ATCase, since isolated catalytic trimers, which cannot exhibit the sequential cooperativity of the model, still exhibit substrate inhibition. Analysis of concerted models (c) shows that a two-state model is inadequate to account for substrate inhibition in ATCase. Further, although unique fits to a three-state model cannot be obtained, because the parameters are highly correlated, several sets of parameter values fit the data well and are in accord with other experimental results. These results indicate that substrate inhibition in ATCase may be the consequence of allostery, and that further experimental investigation is warranted.