The adenine phosphoribosyltransferase from Giardia lamblia has a unique reaction mechanism and unusual substrate binding properties.
Purine phosphoribosyltransferases catalyze the Mg2+ -dependent reaction that transforms a purine base into its corresponding nucleotide. They are present in a wide variety of organisms including plants, mammals, and parasitic protozoa. Giardia lamblia, the causative agent of giardiasis, lacks de novo purine biosynthesis and relies primarily on adenine and guanine phosphoribosyltransferases (APRTase and GPRTase) constituting two independent and essential purine salvage pathways. The APRTase from G. lamblia was cloned and expressed with a 6-His tag at its C terminus and purified to apparent homogeneity. Adenine and alpha-d-5-phosphoribosyl-1-pyrophosphate (PRPP) have K(m) values of 4.2 and 143 microm with a k(cat) of 2.8 s(-1) in the forward reaction, whereas AMP and PP(i) have K(m) values of 87 and 450 microm with a k(cat) of 9.5 x 10(-3) s(-1) in the reverse reaction. Product inhibition studies indicated that the forward reaction follows a random Bi Bi mechanism. Results from the kinetics of equilibrium isotope exchange further verified a random Bi Bi mechanism in the forward reaction. In a mutant enzyme, F25W, with kinetic constants similar to those of the wild type and a tryptophan residue at the adenine binding site, the addition of adenine or AMP to the free mutant enzyme resulted in fluorescence quenching, whereas PRPP caused fluorescence enhancement. The dissociation constants thus estimated are 16.5 microm for adenine, 14.3 microm for AMP, and 83.0 microm for PRPP. PP(i) exerted no detectable effect on the tryptophan fluorescence at all, suggesting a lack of PP(i) binding to the free enzyme. An ordered substrate binding in the reverse reaction with AMP bound first followed by PP(i) is thus postulated.
Sarver
Anne E
AE
Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446, USA.
Wang
Ching C
CC
D000225:Adenine / Q000378:metabolism; D000228:Adenine Phosphoribosyltransferase / Q000737:chemistry*; D000249:Adenosine Monophosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D003001:Cloning, Molecular; D016829:Giardia lamblia / Q000201:enzymology*; D006147:Guanine / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry
D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D006147:Guanine; D014364:Tryptophan; D000228:Adenine Phosphoribosyltransferase; D000225:Adenine