Understanding substrate specificity in human and parasite phosphoribosyltransferases through calculation and experiment

We present molecular dynamics (MD) simulations on two enzymes: a human hypo xanthine-guanine-phosphoribosyltransferase (HGPRTase) and its analogue in t he protozoan parasite Tritichomonas foetus. The parasite enzyme has an addi tional ability to process xanthine as a substrate, making it a hypoxanthine -guanine-xanthine phosphoribosyltransferase (HGXPRTase) [Chin, M. S., and W ang, C. C. (1994) Mol. Biochem. Parasitol. 63 (2), 221-229 (1)]. X-ray crys tal structures of both enzymes complexed to guanine monoribosyl phosphate ( GMP) have been solved, and show only subtle differences in the two active s ites [Eads et al. (1994) Cell 78 (2), 325-334 (2); Somoza et al. (1996) Bio chemistry 35 (22), 7032-7040 (3)]. Most of the direct contacts with the bas e region of the substrate are made by the protein backbone, complicating th e identification of residues significantly associated with xanthine recogni tion. Our calculations suggest that the broader specificity of the parasite enzyme is due to a significantly more flexible base-binding region, and ra tionalize the effect of two mutations, R155E and D163N, that alter substrat e specificity [Munagala, N. R., and Wang, C. C. (1998) Biochemistry 37 (47) , 16612-16619 (4)]. In addition, our simulations suggested a double mutant (D106E/D163N) that might rescue the D163N mutant. This double mutant was ex pressed and assayed, and its catalytic activity was confirmed. Our molecula r dynamics trajectories were also used with a structure-based design progra m, Pictorial Representation Of Free Energy Changes (PROFEC), to suggest par asite-selective derivatives of GMP. Our calculations here successfully rati onalize the parasite-selectivity of two novel inhibitors derived from the c omputer-aided design of Somoza et al. (5) and demonstrate the utililty of P ROFEC in the design of species-selective inhibitors.