Concerted proton transfer mechanism of Clostridium thermocellum ribose-5-phosphate isomerase.
Ribose-5-phosphate isomerase (Rpi) catalyzes the interconversion of D-ribose-5-phosphate and D-ribulose-5-phosphate and plays an essential role in the pentose phosphate pathway and the Calvin cycle of photosynthesis. RpiB, one of the two isoforms of Rpi, is also a potential drug target for some pathogenic bacteria. Clostridium thermocellum ribose-5-phosphate isomerase (CtRpi), belonging to the RpiB family, has recently been employed in the industrial production of rare sugars because of its fast reaction kinetics and narrow substrate specificity. It is known that this enzyme adopts a proton transfer mechanism. It was suggested that the deprotonated Cys65 attracts the proton at C2 of the substrate to initiate the isomerization reaction, and this step is the rate-limiting step. However the elaborate catalytic mechanism is still unclear. We have performed quantum mechanical/molecular mechanical simulations of this rate-limiting step of the reaction catalyzed by CtRpi with the substrate D-ribose. Our results demonstrate that the deprotonated Cys65 is not a stable reactant. Instead, our calculations revealed a concerted proton-transfer mechanism: Asp8, a highly conserved residue in the RpiB family, performs as the base to abstract the proton at Cys65 and Cys65 in turn abstracting the proton of the D-ribose simultaneously. Moreover, we found Thr67 cannot catalyze the proton transfer from O2 to O1 of the D-ribose alone. Water molecule(s) may assist this proton transfer with Thr67. Our findings lead to a clear understanding of the catalysis mechanism of the RpiB family and should guide experiments to increase the catalysis efficiency. This study also highlights the importance of initial protonation states of cysteines.
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