Altering the purine specificity of hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus by structure-based point mutations in the enzyme protein

The hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) fro m Tritrichomonas foetus has been proven to be a target for potential anti-t ritrichomonial chemotherapy. Using a structure-based approach, the base-bin ding region of the active site of this enzyme, which confers unique purine base specificity, was characterized using site-directed mutagenesis. Determ ining the roles of different active-site residues in purine specificity wou ld form the basis for designing specific inhibitors toward the parasitic en zyme. A D163N mutant converts the HGXPRTase into a HGPRTase, which no longe r recognizes xanthine as a substrate, whereas specificities toward guanine and hypoxanthine are unaffected. Apparently, the side-chain carboxyl of Asp 163 forms a hydrogen bond through a water molecule with the C2-carbonyl of xanthine, which constitutes the critical force enabling the enzyme to recog nize xanthine as a substrate. Mutations of Arg155, which orients and stacks the neighboring Tyr156 onto the bound purine base by forming a salt bridge between itself and Glu11, result in drastic increases in the K(m)s for GMP and XMP (but not IMP). This change leads to increased k(cat)s for the forw ard reactions with guanine and xanthine as substrates without affecting the conversion of hypoxanthine to IMP. Thus, the apparent dislocation of Tyr15 6, resulted from mutations of Arg155, bring little effect on the hydrophobi c interactions between Tyr156 and the purine ring. But the forces involved in recognizing the exocyclic C2-substituents of the purine ring, which invo lve the Tyr156 hydroxyl, Ile157 backbone carbonyl, and Asp163 side-chain ca rboxyl, may be weakened by the shifted conformation of the peptide backbone resulted from loss of the Glu11-Arg155 salt bridge. The conserved Lys134 w as proven to be the primary determinant in conferring the specificity of th e enzyme toward 6-oxopurines. By substituting the lysine residue for a seri ne, which can potentially hydrogen bond to either an amino or an oxo-group, we have successfully augmented the purine specificity of the enzyme. The K 134S mutant recognizes adenine in addition to hypoxanthine, guanine, and xa nthine as its substrates. Adenine and hypoxanthine are equivalent substrate s for the mutant enzyme with similar K(m)s of 34.6 and 38.0 mu M, respectiv ely. The catalysis of an adenine phosphoribosyltransferase reaction by this mutant enzyme was further demonstrated by the competitive inhibition of AM P with an estimated K-is of 25.4 mu M against alpha-D-5-phosphoribosyl-pyro phosphate (PRPP) in converting hypoxanthine to IMP. We have thus succeeded in using site-directed mutagenesis to convert T. foetus HGXPRTase into eith er a HGPRTase or a genuine AHGXPRTase.