Jw. Pitera et al., Understanding substrate specificity in human and parasite phosphoribosyltransferases through calculation and experiment, BIOCHEM, 38(32), 1999, pp. 10298-10306
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.