Formation and Reorientation of Glucose 1,6-Bisphosphate in the PMM/PGM Reaction:  Transient-State Kinetic Studies

• Published in: Biochemistry (Easton) 2005-05, Vol.44 (18), p.6831-6836
• Main Authors: Naught, Laura E, Tipton, Peter A
• Format: Article
• Language: English
• Subjects: Animals; Carbon Radioisotopes - metabolism; Catalysis; Crystallography, X-Ray; Glucose-6-Phosphate - analogs & derivatives; Glucose-6-Phosphate - chemistry; Glucose-6-Phosphate - metabolism; Kinetics; Multienzyme Complexes - chemistry; Multienzyme Complexes - metabolism; Muscle, Skeletal - enzymology; Phosphoglucomutase - chemistry; Phosphoglucomutase - metabolism; Phosphorylation; Phosphotransferases (Phosphomutases) - chemistry; Phosphotransferases (Phosphomutases) - metabolism; Pseudomonas aeruginosa - enzymology; Rabbits; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0501380

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 1,6-bisphosphate complex that is not competent to form either glucose 1-phosphate or glucose 6-phosphate directly. We suggest that during the lifetime of this complex the glucose 1,6-bisphosphate intermediate undergoes the 180° 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 1-phosphate or glucose 6-phosphate 14.3 times more frequently than it dissociates from the enzyme.