CRYSTAL-STRUCTURE OF YEAST THYMIDYLATE KINASE COMPLEXED WITH THE BISUBSTRATE INHIBITOR P-1-(5'-ADENOSYL) P-5-(5'-THYMIDYL) PENTAPHOSPHATE (TP(5)A) AT 2.0-ANGSTROM RESOLUTION - IMPLICATIONS FOR CATALYSIS AND AZT ACTIVATION

The crystal structure of yeast thymidylate kinase (TmpK) complexed wit h the bisubstrate inhibitor P-1-(5'-adenosyl) P-5-(5'-thymidyl) pentap hosphate (TP(5)A) was determined at 2.0 Angstrom resolution. In this c omplex, TmpK adopts a closed conformation with a region (LID) of the p rotein closing upon the substrate and forming a helix. The interaction s of TmpK and TP(5)A strongly suggest that arginine 15, which is locat ed in the phosphate binding loop (P-loop) sequence, plays a catalytic role by interacting with an oxygen atom of the transferred phosphoryl group. Unlike other nucleoside monophosphate kinases where basic resid ues from the LID region participate in stabilizing the transition stat e, TmpK lacks such residues in the LID region. We attribute this funct ion to Arg15 of the P-loop. TmpK plays an important role in the phosph orylation of the AIDS prodrug AZT. The structures of TmpK with dTMP an d with AZT-MP [Lavie, A., et al. (1997) Nar. Struct. Biol. 4, 601-604] implicate the movement of Arg15 in response to AZT-MP binding as an i mportant factor in the 200-fold reduced catalytic rate with AZT-MP. Tm pK from Escherichia coli lacks this arginine in its P-loop while havin g basic residues in the LID region. This suggested that, if such a P-l oop movement were to occur in the E. coli TmpK upon AZT-MP binding, it should not have such a detrimental effect on catalysis. This hypothes is was tested, and as postulated, E. coli TmpK phosphorylates AZT-MP o nly 2.5 times slower than dTMP.