The stereochemical course of phospho group transfer catalyzed by rat liver 6-phosphofructo-2-kinase

Adenosine [γ-(S)-¹⁶O,¹⁷O,¹⁸O]triphosphate was used as substrate in the phosphokinase reaction catalyzed by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. The product D-fructose 2,6-[2-¹⁶O,¹⁷O,¹⁸O]bisphosphate was then used as substrate in the alkaline phosphatase-mediated transfer to the phospho groups to (S)-butane-1,3-diol, where the configuration at phosphorus has been determined. Although there was approximately equal transfer by alkaline phosphatase of the labeled 2- and the unlabeled 6-phospho groups, the subsequent assignment of configuration of the chiral phospho group from the 2-position was unambiguous. It was found that 6-phosphofructo-2-kinase proceeds by a pathway that results in net inversion of the configuration at phosphorus. The simplest interpretation of this result is that the phospho group is transferred directly between substrates in a ternary complex by an 'in-line' mechanism not involving a phosphoenzyme intermediate. This conclusion is consistent with the fact that the enzyme cannot be labeled by [γ-³²P]ATP and with steady-state kinetic data that suggest an ordered sequential mechanism. This finding also indicates that the adenine nucleotide and sugar phosphate isotope exchange reactions catalyzed by the enzyme are not relevant to the normal catalytic pathway of the kinase.