Replacement of Val3 in Human Thymidylate Synthase Affects Its Kinetic Properties and Intracellular Stability

Human and other mammalian thymidylate synthase (TS) enzymes have an N-terminal extension of similar to 27 amino acids that is not present in bacterial TSs. The extension, which is disordered ill all reported crystal Structures of TSs, has been considered to play a primary role in Protein turnover but not In catalytic activity. Ill mammalian cells the variant V3A has,I half-life similar to that of wild-type human TS (wt hTS) while V3T is much more stable, V3L, V3F, and V3Y have half-lives approximately half of that for wt hTS. Catalytic turnover rates for most Val3 mutants are only slightly diminished, as expected. However, two Y I, I I I mutants, V3L and V3F, have strongly compromised dUMP binding, with values increased 1 factors of 47 and 58, respectively. For V3L, this observation can be explained by stabilization of the inactive conformation of the loop of residues IS 1-197, which prevents Substrate binding. Ill the crystal Structure Of V3L, electron density corresponding to a leucine residue is present In a position that stabilizes the loop of residues 18 1 - 197 in the inactive conformation. Since this density Is not observed in Other Mutants and all other leucine residues are ordered ill this Structure, It is likely that this density represents Leu3. In the crystal Structure of a V3F.FdUM P binary complex, the nucleotide is bound in in alternative mode to that proposed for the catalytic complex, indicating that the high K-m.app Value IS caused not by stabilization of the Inactive conformer but by substrate binding, ill it nonproductive, Inhibitory site. These observations show that (lie N-terminal extension affects the conformational State of the hTS catalytic region. Each of the mechanisms leading to the high k(m.app) Values can be exploited to facilitate design of compounds acting its allosteric inhibitors of hTS,