Abstract：
:Despite many studies suggesting the role of enolpyruvate as a bound intermediate in the pyruvate kinase reaction, direct evidence for it has been lacking. By use of a combination of chemical trapping and isolation of a derivative, significant amounts of enzyme-bound enolpyruvate have now been demonstrated. The method distinguishes enolpyruvate. It is based on reaction of bromine with enolpyruvate in acid, derivatization of formed bromopyruvate with thionitrobenzoate, and resolution by reversed-phase HPLC of the thioether derivative. As little as 10 pmol of the thioether derivative could be quantitated reliably. With this method, the internal equilibria, including the E.ATP.enolpyruvate intermediate, have been determined. Enzyme-enolpyruvate concentration was shown to be pH-dependent. Phosphoenolpyruvate also reacts with bromine to form bromopyruvate. To quantitate enolpyruvate specifically in a background of phosphoenolpyruvate, advantage was taken of phosphoenolpyruvate's much greater stability in acid. When bromide/was added 10 min after the acid quench, ketonization of enolpyruvate was complete, and only phosphoenolpyruvate was measured. Enolpyruvate is thus determined by difference between the bromopyruvate measured with and without delayed bromine addition.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Seeholzer SH,Jaworowski A,Rose IAdoi
10.1021/bi00217a022subject
Has Abstractpub_date
1991-01-22 00:00:00pages
727-32issue
3eissn
0006-2960issn
1520-4995journal_volume
30pub_type
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