Crystal structure of Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase with XMP, pyrophosphate, and two Mg(2+) ions bound: insights into the catalytic mechanism.
The crystal structure of the Toxoplasma gondii hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-xanthosine 5'-monophosphate (XMP)-pyrophosphate-Mg(2+) ternary complex has been determined at 1. 60 A resolution. This biproduct, post-transition state structure is of a T. gondii HGPRT mutant (Asp150Ala or D150A). The D150A mutant has reduced activity (k(cat) lower by 11-, 296-, and 8.6-fold for hypoxanthine, guanine, and xanthine, respectively) compared to wild-type T. gondii HGPRT. The Michaelis constants for purine bases are altered only slightly, whereas those for alpha-D-5-phosphoribosyl 1-pyrophosphate (PRPP) are lower by approximately 6.5-fold. The ternary complex crystallizes in space group C222(1) (a = 55.21 A, b = 112.25 A, and c = 144.28 A) with two subunits in the asymmetric unit; the HGPRT tetramer is completed by the application of 2-fold crystallographic symmetry. All active sites contain XMP ¿bound in a fashion similar to that of the guanosine 5'-monophosphate (GMP) and inosine 5'-monophosphate (IMP) complexes reported in the preceding article [Héroux, A., et al. (1999) Biochemistry 38, 14485-14494]¿, pyrophosphate, and two Mg(2+) ions. Each Mg(2+) ion is octahedrally coordinated by two terminal pyrophosphate oxygen atoms and several ordered water molecules. This structure shows how HGPRT uses two Mg(2+) ions to orient and activate the pyrophosphate moiety of PRPP for attack by a purine base, and why mutation in humans of the residue corresponding to Asp206, the only HGPRT amino acid that directly contacts the Mg(2+) ions, causes Lesch-Nyhan syndrome (HGPRT(Kinston), D193N). The Leu78-Lys79 peptide bond in the active site adopts the cis configuration, which it must to bind PRPP or pyrophosphate. The contribution of cis-trans isomerization of this peptide bond to the energetics of substrate binding and product release is discussed. A comprehensive description of the HGPRT reaction mechanism is also proposed.
Héroux
A
A
Drug Discovery Division, Southern Research Institute, Birmingham, Alabama 35205, USA.
White
E L
EL
Ross
L J
LJ
Davis
R L
RL
Borhani
D W
DW
D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000235:genetics; D011756:Diphosphates / Q000737:chemistry; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012265:Ribonucleotides / Q000737:chemistry; D014122:Toxoplasma / Q000201:enzymology* / Q000235:genetics; D019820:Xanthine
D017931:DNA Primers; D011756:Diphosphates; D012265:Ribonucleotides; D019820:Xanthine; C011141:xanthosine monophosphate; D007041:Hypoxanthine Phosphoribosyltransferase; D008274:Magnesium