Formation and reorientation of glucose 1,6-bisphosphate in the PMM/PGM reaction: Transient-state kinetic studies

The interconversion of glucose 1-phosphate and glucose 6-phosphate, catalyzed by Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase, has been studied by transient-state kinetic techniques. Glucose 1,6-bisphosphate is formed as an intermediate in the reaction, but an obligatory step in the catalytic cycle appears to be the formation of an enzyme-glucose 4,6-bisphosphate complex that is not competent to form either glucose I-phosphate or glucose 6-phosphate directly. We suggest that during the lifetime of this complex the glucose 1,6-bisphosphate intermediate undergoes the 180 degrees reorientation that is required for completion of the catalytic cycle. The formation of glucose 1,6-bisphosphate from glucose 1-phosphate is in rapid equilibrium relative to the rest of the reaction, where K-eq = 0.14. In the opposite direction, glucose 1,6-bisphosphate is formed from glucose 6-phosphate with a rate constant of 12 s(-1), and the reverse step occurs with a rate constant of 255 s(-1). The interconversion of the productive and nonproductive glucose 1,6-bisphosphate complexes occurs with a rate constant of 64 s(-1) in one direction and 48 s-1 in the other direction. Glucose 1,6-bisphosphate remains associated with the enzyme during reorientation. Isotope trapping studies indicate that it partitions to form glucose I-phosphate or glucose 6-phosphate 14.3 times more frequently than it dissociates from the enzyme.