Perturbation of yeast 3-phosphoglycerate kinase reaction mixtures with ADP: transient kinetics of formation of ATP from bound 1,3-bisphosphoglycerate.
3-Phosphoglycerate kinase (PGK) is the first ATP-producing enzyme in glycolysis: ADP + 1,3-bisphosphoglycerate (bPG) <--> ATP + 3-phosphoglycerate (PG). Whereas extensive studies have been carried out on its structure, there is less information about its reaction pathway, which is usually studied in the reverse direction because of the instability of bPG. We studied the transients of the PGK reaction by chemical sampling in a rapid quench flow apparatus, using [gamma-(32)P]ATP, in 30% methanol at 4 degrees C to decrease k(cat). There were two types of experiment, both at low PG concentrations to prevent bPG release. In the first, reaction mixtures were quenched in acid at different times (from 4 ms) and the bPG concentrations were determined. This type gave information about the ATP binding and phospho-transfer steps. In the second, PGK reaction mixtures at equilibrium were perturbed by the injection of ADP, the new mixtures aged for different times and quenched in acid, and the bPG concentrations were determined. This gave information about the kinetics of the binding of ADP to a PGK intermediate. The data from the two types of experiments were fitted to simple schemes and then treated together by a global fitting procedure using a five-step pathway, deduced from previous structural studies. Under our conditions, it appears that (1) a binary PGK.bPG complex is an important intermediate on the reaction pathway, i.e., that ADP is released before bPG, (2) ADP binds to a "closed" conformation in the PGK.bPG complex, and (3) the PGK reaction can be studied in the physiologically important direction without having to handle bPG.
Geerlof
Arie
A
EMBL Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany.
Travers
Franck
F
Barman
Tom
T
Lionne
Corinne
C
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D004163:Diphosphoglyceric Acids / Q000378:metabolism*; D005988:Glyceric Acids / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology
D004163:Diphosphoglyceric Acids; D005988:Glyceric Acids; C015891:glycerate 1,3-biphosphate; D000244:Adenosine Diphosphate; C005156:3-phosphoglycerate; D000255:Adenosine Triphosphate; D010735:Phosphoglycerate Kinase