| Abstrakt: |
The influence of pH on the activity of purified pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Trypanosoma brucei has been studied. The Km for the coenzyme ADP is pH-dependent and shows the involvement of a dissociable group on the free enzyme with a pKa of 6.5-6.7. The cooperative interaction of the multiple phosphoenolpyruvate (PEP) binding sites is independent of pH in the range of 5.7-7.8. Variation of the Vmax value with pH indicates the presence of a dissociated group (pKa 6.2-6.3) and of an undissociated group (pKa 7.5-7.6) in the enzyme-substrate complex. A doubly dissociated phosphate group on PEP is shown to be essential by the effects of pH on the S0.5 value for this substrate, as is an undissociated enzyme group with a pKa in the range 6.7-7.0. It is shown that PEP and frucotse-1,6-diphosphate (FDP) act entirely in conjunction in allosterically activating the enzyme, FDP, the heterotropic effector, decreases the interaction between PEP binding sites at low concentration, and decreases the S0.5 value for PEP at higher concentration. A model for the interaction of the enzyme with its substrates is discussed. |
