Mechanism of 1,3-bisphosphoglycerate transfer from phosphoglycerate kinase to glyceraldehyde-3-phosphate dehydrogenase.
1. The kinetics of 1,3-bisphosphoglycerate binding to glyceraldehyde-3-phosphate dehydrogenase have been examined by stopped-flow techniques in the absence and presence of phosphoglycerate kinase, using enzyme concentrations in the range 0.5-40 microM. Rate and equilibrium constant estimates for the interaction of the ligand with the two enzymes are reported. 2. The kinetics of ligand transfer from the binary complex of bisphosphoglycerate and phosphoglycerate kinase to the binary complex of NAD+ and glyceraldehyde-3-phosphate dehydrogenase conform excellently to the predictions of a standard free-diffusion mechanism and exhibit no detectable contributions from a mechanism of direct (channelized) transfer of bisphosphoglycerate between the two enzymes. 3. Previously reported evidence that the binary complex of bisphosphoglycerate and phosphoglycerate kinase may act (in the presence of NADH) as a substrate for glyceraldehyde-3-phosphate dehydrogenase according to Michaelis-Menten kinetics is based on a misinterpretation of the experimental observations that can be attributed to neglect of the autocatalytic effect of NAD+ produced during the reaction. Experiments performed under conditions where the autocatalytic effect of NAD+ is eliminated provide clear evidence that the kinetics of utilization of the kinase-bisphosphoglycerate complex for enzymic NADH reduction are consistent with prior dissociation of the complex according to a free-diffusion mechanism of metabolite transfer and incompatible with a mechanism of direct metabolite transfer. 4. A kinetic argument is presented which renders implausible the very idea that direct metabolite transfer between 'soluble' consecutive enzymes in metabolic pathways may offer any catalytic advantages in comparison to metabolite transfer by free diffusion. A mechanism of direct metabolite transfer seems intuitively attractive only because one tends to disregard the diffusional processes required to bring the consecutive enzymes together and to separate them when the transfer has been completed. Direct metabolite transfer would be expected to be catalytically advantageous only in tightly bound multienzyme complexes showing no kinetically significant tendency to dissociate. 5. It is concluded that mechanisms of direct metabolite transfer have not been convincingly demonstrated to apply, nor are they likely to apply, between 'soluble' consecutive enzymes in metabolic pathways, at least not in the glycolytic sequence of reactions.
Kvassman
J
J
Avdelningen för Biokem, Lunds Universitet, Sweden.
Pettersson
G
G
D000818:Animals; D002384:Catalysis; D004163:Diphosphoglyceric Acids / Q000378:metabolism*; D005412:Flatfishes; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D010735:Phosphoglycerate Kinase / Q000378:metabolism*
D004163:Diphosphoglyceric Acids; D009243:NAD; C015891:glycerate 1,3-biphosphate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010735:Phosphoglycerate Kinase