EFFECTS OF ASSEMBLY AND MUTATIONS OUTSIDE THE ACTIVE-SITE ON THE FUNCTIONAL PH-DEPENDENCE OF ESCHERICHIA-COLI ASPARTATE-TRANSCARBAMYLASE

Electrostatics are central to the function and regulation of Escherich ia coil aspartate transcarbamylase, and modeling has suggested that lo ng range electrostatic effects are likely to be important (Glackin, M. P., McCarthy, M.P., Mallikarachchi, D., Matthew, J.B., and Allewell, N .M. (1989) Proteins Struct. Funct. Genet. 5, 66-77; Oberoi, H., Trikha , J., Yuan, X., and Allewell, N. M. (1995) Proteins Struct. Funct. Gen et., in press). To investigate this possibility from an experimental s tandpoint, we have examined the effects both of assembly and of removi ng ionizable and polar side chains outside the active site (Glu-50, Ty r-165, and Tyr-240) on the pH dependence of the kinetic parameters of aspartate transcarbamylase. The holoenzyme (c(6)r(6)) assembles from t hree regulatory dimers (r(2)) and two catalytically active trimers (c( 3)). pH dependences of the enzyme kinetic parameters suggest that the mechanisms of productive binding of L-Asp to the binary complexes of t he catalytic subunit (c(3)) and holoenzyme (c(6)r(6)) with carbamyl ph osphate are different. In contrast, the Michaelis complex appears simi lar for both c(3) and c(6)r(6), except for pK shifts of similar to 1 p H unit. Results also indicate that the catalytic mechanism of the holo enzyme does not involve reverse protonation, as has recently been prop osed for the catalytic trimer (Turnbull, J. L., Waldrop, G. L., and Sc hachman, H. K. (1992) Biochemistry 31, 6562-6569). The tyrosines at po sitions 165 and 240 are part of a cluster of interactions that links t he catalytic subunits in the T state (the c1:c4 interface) and which i s disrupted in the T-->R transition. The effects of mutating the two T yr residues are quite different: Y240F has higher than wild-type activ ity and affinity over the entire pH range, while Y165F has activity an d affinity an order of magnitude lower than wild-type. Removal of the regulatory subunits from Y165F increases activity and affinity and res tores the pH dependence of the wildtype catalytic subunit. Like Y165F, E50A has low activity and affinity over the entire pH range. Linkage analysis indicates that there is long range energetic coupling among t he active site, the c:r subunit interfaces, and residue Y165. The subs tantial quantitative difference between Y165F and Y240F, both of which are at the cl:c4 interface about 14-16 if from the closest active sit e, demonstrates specific path dependence, as opposed to general distan ce dependence, of interactions between this interface and the active s ite.