| Abstract
| - Adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) catalyzes the reversible phosphoribosylation of adenine from α-d-5-phosphoribosyl-1-pyrophosphate (PRPP) to form AMP and PPi. Three-dimensional structures of the dimeric APRT enzyme from Leishmania donovani (LdAPRT) bear manysimilarities to other members of the type 1 phosphoribosyltransferase family but do not reveal the structuralbasis for catalysis (Phillips, C. L., Ullman, B., Brennan, R. G., and Hill, C. P. (1999) EMBO J.18,3533−3545). To address this issue, a steady state and transient kinetic analysis of the enzyme was performedin order to determine the catalytic mechanism. Initial velocity and product inhibition studies indicatedthat LdAPRT follows an ordered sequential mechanism in which PRPP is the first substrate to bind andAMP is the last product to leave. This mechanistic model was substantiated by equilibrium isotope exchangeand fluorescence binding studies, which provided dissociation constants for the LdAPRT−PRPP andLdAPRT−AMP binary complexes. Pre-steady-state kinetic analysis of the forward reaction revealed aburst in product formation indicating that phosphoribosyl transfer proceeds rapidly relative to some rate-limiting product release event. Transient fluorescence competition experiments enabled measurement ofrates of binary complex dissociation that implicated AMP release as rate-limiting for the forward reaction.Kinetics of product ternary complex formation were evaluated using the fluorophore formycin AMP andestablished rate constants for pyrophosphate binding to the LdAPRT−formycin AMP complex. Takentogether, these data enabled the complete formulation of an ordered bi−bi kinetic mechanism for LdAPRTin which all of the rate constants were either measured or calculated.
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