The role of phosphate in a multistep enzymatic reaction.

摘要

Many enzymes operate on substrates containing phosphate groups. Although they are often remote from the reactive center, phosphates are essential for recognition but play no obvious catalytic function. Previous studies provided evidence for the involvement of the phosphodianion binding energy in transition state formation of several mechanistically unrelated enzymes. However, the employed model systems have been limited to the enzymes which are (1) highly proficient catalysts from eukaryotic sources, (2) independent of cofactors, and (3) monofunctional. Although two multistep model reactions involving reactive intermediate(s) have been considered, no previous systematic investigation has examined how the phosphodianion binding energy changes along the reaction coordinate. The current study aims to address these major gaps by investigating the reaction catalyzed by 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), a bifunctional metal-dependent enzyme largely restricted to microbial sources. Accordingly, we report (1) the synthesis and kinetic characterization of DXR's substrate and intermediate as well as their counterparts lacking the phosphoryloxymethyl groups, (2) kinetics of activation of the truncated substrate and intermediate turnover by phosphite dianion, (3) kinetic isotope effects in the respective reactions, and (4) X-ray crystallographic characterization of DXR in complex with the substrate in pieces. Our major findings indicate (1) suboptimal utilization of phosphodianion binding energy by DXR, (2) differential degrees of phosphodianion-provided stabilization of individual kinetic barriers to the reaction, (3) the role of the phosphodianion in disfavoring intermediate release and in modulation of the on-enzyme isomerization equilibrium, (4) rate limitation by physical steps when the covalent linkage to phosphate is severed. In summary, this investigation broadens our understanding of the role of nonreacting phosphoryl groups in enzymatic reactions.