Transient and equilibrium kinetic studies on yeast 3-phosphoglycerate kinase. Evidence that an intermediate containing 1,3-bisphosphoglycerate accumulates in the steady state.
The structure of the key glycolytic enzyme 3-phosphoglycerate kinase (PGK) is known in detail, but there is little information on its reaction pathway. We have studied its equilibrium and transient kinetics in the direction of 1,3-bisphosphoglycerate (1,3-bis-P-glycerate) production: ATP + 3-P-glycerate<==>ADP + 1,3-bis-P-glycerate. We devised a sensitive method for following this production. PGK is mixed with 3-P-glycerate and [gamma-32P]ATP in a rapid flow quench apparatus. The reaction mixtures are aged for 4 ms or more and then quenched in acid in which any [1-32P]-1,3-P-glycerate decomposes to 3-P-glycerate and 32Pi, which is determined specifically. The Pi reflects accurately the 1,3-bis-P-glycerate in the original reaction mixture, and the kcat obtained is identical to that obtained by the conventional linked assay method with glyceraldehyde-3-phosphate dehydrogenase. This does not support the postulate of a rapid direct transfer of the 1,3-bis-P-glycerate between the kinase and the dehydrogenase [Srivastava, D. K., & Bernhard, S. A. (1986) Science 234, 1081-1086]. We fitted our data to a simple scheme with the formation of binary complexes, the interconversion of substrates to products via ternary complexes, and the release of products. Because of the high turnover of PGK, the work was carried out under cryoenzymic conditions with 40% ethylene glycol in the buffer. The glycol decreased kcat from 80 to 8.5 s-1 (pH 7.5, 4 degrees C), but the Km for 3-P-glycerate and ATP and the equilibrium constants in the scheme were little affected. We carried out two types of experiment.(ABSTRACT TRUNCATED AT 250 WORDS)[1]References
- Transient and equilibrium kinetic studies on yeast 3-phosphoglycerate kinase. Evidence that an intermediate containing 1,3-bisphosphoglycerate accumulates in the steady state. Schmidt, P.P., Travers, F., Barman, T. Biochemistry (1995) [Pubmed]
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