Altering the purine specificity of hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus by structure-based point mutations in the enzyme protein
Nr. Munagala et Cc. Wang, Altering the purine specificity of hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus by structure-based point mutations in the enzyme protein, BIOCHEM, 37(47), 1998, pp. 16612-16619
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.