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J. Biol. Chem., Vol. 277, Issue 42, 39973-39980, October 18, 2002

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The Adenine Phosphoribosyltransferase from Giardia lamblia Has a Unique Reaction Mechanism and Unusual Substrate Binding Properties^*

Anne E. Sarver and Ching C. Wang^§

From the Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446

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 -D-5-phosphoribosyl-1-pyrophosphate (PRPP) have K_m values of 4.2 and 143 µM with a k_cat of 2.8 s¹ in the forward reaction, whereas AMP and PP_i have K_m values of 87 and 450 µM with a k_cat of 9.5 × 10³ s¹ 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 µM for adenine, 14.3 µM for AMP, and 83.0 µM 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF378363.

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				W. Shi, A. E. Sarver, C. C. Wang, K. S. E. Tanaka, S. C. Almo, and V. L. Schramm Closed Site Complexes of Adenine Phosphoribosyltransferase from Giardia lamblia Reveal a Mechanism of Ribosyl Migration J. Biol. Chem., October 11, 2002; 277(42): 39981 - 39988. [Abstract] [Full Text] [PDF]