Kinetic mechanism of adenine phosphoribosyltransferase from Leishmania donovani.
Adenine phosphoribosyltransferase (APRT, EC 2.4.2.7) catalyzes the reversible phosphoribosylation of adenine from alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) to form AMP and PP(i). Three-dimensional structures of the dimeric APRT enzyme from Leishmania donovani (LdAPRT) bear many similarities to other members of the type 1 phosphoribosyltransferase family but do not reveal the structural basis 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 performed in order to determine the catalytic mechanism. Initial velocity and product inhibition studies indicated that LdAPRT follows an ordered sequential mechanism in which PRPP is the first substrate to bind and AMP is the last product to leave. This mechanistic model was substantiated by equilibrium isotope exchange and fluorescence binding studies, which provided dissociation constants for the LdAPRT-PRPP and LdAPRT-AMP binary complexes. Pre-steady-state kinetic analysis of the forward reaction revealed a burst in product formation indicating that phosphoribosyl transfer proceeds rapidly relative to some rate-limiting product release event. Transient fluorescence competition experiments enabled measurement of rates 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 and established rate constants for pyrophosphate binding to the LdAPRT-formycin AMP complex. Taken together, these data enabled the complete formulation of an ordered bi-bi kinetic mechanism for LdAPRT in which all of the rate constants were either measured or calculated.
Bashor
Caleb
C
Department of Biochemistry and Molecular Biology, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, Oregon 97201-3098, USA.
Denu
John M
JM
Brennan
Richard G
RG
Ullman
Buddy
B
D000228:Adenine Phosphoribosyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D007700:Kinetics; D007893:Leishmania donovani / Q000201:enzymology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D000228:Adenine Phosphoribosyltransferase