IS SUBSTRATE-INHIBITION A CONSEQUENCE OF ALLOSTERY IN ASPARTATE-TRANSCARBAMYLASE

Aspartate transcarbamylase (ATCase) is a highly regulated, multisubuni t enzyme that catalyzes the first regulated step in pyrimidine biosynt hesis. Although ATCase exhibits strong substrate inhibition (the reduc tion of enzyme activity at high substrate concentrations), the mechani sm of substrate inhibition has not been investigated. At the molecular level, substrate inhibition may result either from local events at th e active site or from global or specific long-range allosteric effects . We have compared the results of fitting kinetic data to several mode ls: (a) a semi-empirical steady-state kinetic model that includes coop erative substrate binding (described by a Hill coefficient) and partia l uncompetitive substrate inhibition, (b) a nested allosteric model de veloped to analyze substrate inhibition of the ATPase activity of GroE L, an enzyme with a quaternary structure analogous to ATCase (O. Yifra ch and A. Horovitz, Biochemistry, 34 (1995) 5303), and (c) purely conc erted models, including a model originally proposed by Monod et al. (J . Monod, J. Wyman and J.P. Changeux, J. Mol. Biol., 12 (1965) 88). Mod el (a) is the first kinetic equation for ATCase that both fits the dat a and returns physically realistic values for all parameters, but it i s a modified Hill equation and thus returns little or no molecular mec hanistic information. The nested allosteric model (b), which assumes c oncerted cooperativity within each catalytic trimer of ATCase and sequ ential cooperativity between trimers, is unlikely to be the correct mo del for ATCase, since isolated catalytic trimers, which cannot exhibit the sequential cooperativity of the model, still exhibit substrate in hibition. Analysis of concerted models (c) shows that a two-state mode l is inadequate to account for substrate inhibition in ATCase. Further , although unique fits to a three-state model cannot be obtained, beca use the parameters are highly correlated, several sets of parameter va lues fit the data well and are in accord with other experimental resul ts. These results indicate that substrate inhibition in ATCase may be the consequence of allostery, and that further experimental investigat ion is warranted.