NMR spectroscopic analysis of the first two steps of the pentose-phosphatepathway elucidates the role of 6-phosphogluconolactonase

The pentose-phosphate pathway provides reductive power and nucleotide precu rsors to the cell through oxidative and nonoxidative branches, respectively . 6-Phosphogluconolactonase is the second enzyme of the oxidative branch an d catalyzes the hydrolysis of 6-phosphogluconolactones, the products of glu cose 6-phosphate oxidation by glucose-6-phosphate dehydrogenase. The role o f 6-phosphogluconolactonase was still questionable, because 6-phosphoglucon olactones were believed to undergo rapid spontaneous hydrolysis. In this wo rk, nuclear magnetic resonance spectroscopy was used to characterize the ch emical scheme and kinetic features of the oxidative branch. We show that 6- phosphogluconolactones have in fact a nonnegligible life. time and are high ly electrophilic compounds. The delta form (1-5) of the lactone is the only product of glucose 6-phosphate oxidation. Subsequently, it leads to the ga mma form (1-4) by intramolecular rearrangement. However, only the delta for m undergoes spontaneous hydrolysis, they form being a "dead end" of this br anch. The delta form is the only substrate for 6-phosphogluconolactonase. T herefore, 6-phosphogluconolactonase activity accelerates hydrolysis of the delta form, thus preventing its conversion into the gamma form. Furthermore , 6-phosphogluconolactonase guards against the accumulation of 5-6-phosphog luconolactone, which may be toxic through its reaction with endogenous cell ular nucleophiles. Finally, the difference between activity of human, Trypa nosoma brucei, and Plasmodium falciparum 6-phosphogluconolactonases is repo rted and discussed.