The role for an invariant aspartic acid in hypoxanthine phosphoribosyltransferases is examined using saturation mutagenesis, functional analysis, andX-ray crystallography

The role of an invariant aspartic acid (Asp137) in hypoxanthine phosphoribo syltransferases (HPRTs) was examined by site-directed and saturation mutage nesis, Functional analysis, and X-ray crystallography using the HPRT from T rypanosoma cruzi. Alanine substitution (D137A) resulted in a 30-fold decrea se of k(cat), suggesting that Asp137 participates in catalysis. Saturation mutagenesis was used to generate a library of mutant HPRTs with random subs titutions at position 137, and active enzymes were identified by complement ation of a bacterial purine auxotroph. Functional analyses of the mutants, including determination of steady-state kinetic parameters and pH-rate depe ndence, indicate that glutamic acid or glutamine can replace the wild-type aspartate. However, the catalytic efficiency and pH-rate profile for the st ructural isosteric mutant, D137N, were similar to the D137A mutant. Crystal structures of four of the mutant enzymes were determined in ternary comple x with substrate ligands. Structures of the D137E and D137Q mutants reveal potential hydrogen bonds, utilizing several bound water molecules in additi on to protein atoms, that position these side chains within hydrogen bond d istance of the bound purine analogue, similar in position to the aspartate in the wild-type structure. The crystal structure of the D137N mutant demon strates that the Asn137 side chain does not form interactions with the puri ne substrate but instead forms novel interactions that cause the side chain to adopt a nonfunctional rotamer. The results from these structural and fu nctional analyses demonstrate that HPRTs do not require a general base at p osition 137 for catalysis. Instead, hydrogen bonding sufficiently stabilize s the developing partial positive charge at the N7-atom of the purine subst rate in the transition-state to promote catalysis.