The Carboxypeptidase-dependent Inactivation of Thymidylate Synthetase

Complete Restoration of Activity to Inactive Mutants of Escherichia coli Thymidylate Synthase: Evidence that E. coli Thymidylate Synthase is a Half-the-Sites Activity Enzyme

“…It was hoped that only the active TS heterodimers would form a ternary complex with [,2P]FdUMP and CH.HjPteGlu, as opposed to the inactive mutants, so that the number of functionally active TS sites of heterodimer could be established by the extent of migration of the radioactive ternary complexes. This thesis is based on the finding of Aull et al (1974) that each of the active subunits forms a ternary complex with FdUMP and CHiHjPteGlu, and the enzyme dimer containing one FdUMP migrates slower than that containing two FdUMP.…”

“…Charge differences do not appear to be involved since these mutants when subjected to isoelectric focusing do not show any significant differences in mobility (data not presented). If it is assumed that complex formation with FdUMP and CH2H4PteGlu yields a faster migrating species depending on whether one or two FdUMPs are bound/dimer (lane 12) (Aull et al, 1974), it can be assumed that the faster moving labeled band in lane 15 is the result of a single FdUMP being bound/dimer of (R126E)-(C146W). This band was not seen with Ci46W (lanes 5 and 6) and only weakly in RmE (lanes 2 and 3), which suggests that the strong radioactive band in lane 15 is in all likelihood due to the enhanced binding of FdUMP to the active subunit of the mixed heterodimer (R126E) (Ci 46W).…”

“…The crystal structures of both Lactobacillus casei (Hardy et al, 1986) and E. coli TS (Matthews et al, 1990 a,b;Montfort et al, 1990) reveal that the enzyme's two identical subunits are held together by a backbone of about 30 amino acid residues with the active site in each subunit widely separated from one another despite the marked change in conformation that occurs on substrate or analogue binding. This being the case, each site should be equally active although a considerable body of evidence exists suggesting either that the two active sites contribute unequally to the enzyme's catalytic efficiency {kcx) or that only one of the two sites is active (Aull et al, 1974;Leary et al, 1975;Galivan et al, 1976a,b;Danenberg & Danenberg, 1979;Beaudette et al, 1980). Paradoxically, other studies suggest that both sites contribute equally to enzyme activity (Plese & Dunlap, 1977), particularly those involving subunit complementation wherein specific inactive mutants of L. casei TS were denatured together with urea and then renaturated by dilution, resulting in the restoration of enzyme activity (Perry et al, 1992;Pookanjanatavip et al, 1992;Carreras et al, 1994).…”

Characterization of a novel form of thymidylate synthase: A heterodimer isolated after specific chemical modification of the immobilized native enzyme

“…The relative proximity of the two active sites at the interface of the two identical subunits also makes this contention plausible. The delicate interplay of the two active sites is further illustrated by carboxypeptidase A studies (Loeble & Dunlap, 1972;Aull et al, 1974a;Cisneros et al, 1993), which demonstrate that the removal of the carboxyl-terminal valine from one subunit results in a form of negative cooperativity expressed as the complete loss of dTMP synthesis activity but the retention of ternary complex formation at one active site. Why does the single-site dimer form produced in this study exhibit positive cooperativity while the heterodimer resulting from carboxypeptidase A treatment evidences negative cooperativity?…”

“…The fact that the dimeric protein does not dissociate to monomeric species under native conditions formed the basis for two different approaches leading to the generation of interesting heterodimeric species of the enzyme which have been employed to probe the nature and basis of subunit communication. First, and perhaps the most dramatic illustration of subunit communication in this enzyme, was the approach based on the finding that the carboxypeptidase A-dependent removal of the C-terminal residue from just one of the two identical subunits corresponds to the loss of dTMP synthesis activity at both enzymic active sites (Loeble & Dunlap, 1972;Aull et al, 1974a). Even more surprisingly, the subtly modified heterodimer resulting from limited treatment of thymidylate synthase with carboxypeptidase A continues to catalyze the formation and decay of covalent binary and ternary complexes, but these events are restricted to just one of the two subunits of the enzyme (Cisneros et al, 1993).…”