TY - JOUR
T1 - Pyrophosphate interactions at the transition states of plasmodium falciparum and human orotate phosphoribosyltransferases
AU - Zhang, Yong
AU - Schramm, Vern L.
PY - 2010/6/30
Y1 - 2010/6/30
N2 - Orotate phosphoribosyltransferases from Plasmodium falciparum and human sources (PfOPRT and HsOPRT) use orotidine as a slow substrate in the pyrophosphorolysis reaction. With orotidine, intrinsic kinetic isotope effects (KIEs) can be measured for pyrophosphorolysis, providing the first use of pyrophosphate (PPi) in solving an enzymatic transition state. Transition-state structures of PfOPRT and HsOPRT were solved through quantum chemical matching of computed and experimental intrinsic KIEs and can be compared to transition states solved with pyrophosphate analogues as slow substrates. PfOPRT and HsOPRT are characterized by late transition states with fully dissociated orotate, well-developed ribocations, and weakly bonded PPi nucleophiles. The leaving orotates are 2.8 Å distant from the anomeric carbons at the transition states. Weak participation of the PPi nucleophiles gives C1′-O PPi bond distances of approximately 2.3 Å. These transition states are characterized by C2′-endo ribosyl pucker, based on the β-secondary [2′-3H] KIEs. The geometry at the 5′-region is similar for both enzymes, with C3′-C4′-C5′- O5′ dihedral angles near -170°. These novel phosphoribosyltransferase transition states are similar to but occur earlier in the reaction coordinate than those previously determined with orotidine 5′-monophosphate and phosphonoacetic acid as substrates. The similarity between the transition states with different substrate analogues supports similar transition state structures imposed by PfOPRT and HsOPRT even with distinct reactants. We propose that the transition state similarity with different nucleophiles is determined, in part, by the geometric constraints imposed by the catalytic sites.
AB - Orotate phosphoribosyltransferases from Plasmodium falciparum and human sources (PfOPRT and HsOPRT) use orotidine as a slow substrate in the pyrophosphorolysis reaction. With orotidine, intrinsic kinetic isotope effects (KIEs) can be measured for pyrophosphorolysis, providing the first use of pyrophosphate (PPi) in solving an enzymatic transition state. Transition-state structures of PfOPRT and HsOPRT were solved through quantum chemical matching of computed and experimental intrinsic KIEs and can be compared to transition states solved with pyrophosphate analogues as slow substrates. PfOPRT and HsOPRT are characterized by late transition states with fully dissociated orotate, well-developed ribocations, and weakly bonded PPi nucleophiles. The leaving orotates are 2.8 Å distant from the anomeric carbons at the transition states. Weak participation of the PPi nucleophiles gives C1′-O PPi bond distances of approximately 2.3 Å. These transition states are characterized by C2′-endo ribosyl pucker, based on the β-secondary [2′-3H] KIEs. The geometry at the 5′-region is similar for both enzymes, with C3′-C4′-C5′- O5′ dihedral angles near -170°. These novel phosphoribosyltransferase transition states are similar to but occur earlier in the reaction coordinate than those previously determined with orotidine 5′-monophosphate and phosphonoacetic acid as substrates. The similarity between the transition states with different substrate analogues supports similar transition state structures imposed by PfOPRT and HsOPRT even with distinct reactants. We propose that the transition state similarity with different nucleophiles is determined, in part, by the geometric constraints imposed by the catalytic sites.
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U2 - 10.1021/ja102849w
DO - 10.1021/ja102849w
M3 - Article
C2 - 20527751
AN - SCOPUS:77953892029
SN - 0002-7863
VL - 132
SP - 8787
EP - 8794
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 25
ER -