Phosphorylation of pyruvate by the pyruvate kinase reaction and reversal of glycolysis in a reconstructed system.

Lardy and Ziegler (1) obtained synthesis of phosphoenol pyruvate from pyruvate and adenosine triphosphate by reversal of the pyruvate kinase reaction. A possible mechanism was thus demonstrated for the initial step in the synthesis of glycogen from pyruvate. The amounts of phosphoenol pyruvate synthesized were, however, small, and it is rather widely held that reversal of the pyruvate kinase reaction leading to net synthesis of phosphoenol pyruvate in viva is insignificant (Z-5). It is the purpose of this paper to report the synthesis of relatively large amounts of phosphoenol pyruvatel in an enzymatic system similar to that of Lardy and Ziegler. They used muscle extracts to generate ATP by the oxidative phosphorylation of glyceraldehyde 3-phosphate; the enolase reaction was blocked with fluoride. It was found that substitution of the muscle extracts by purified glycolytic enzymes resulted in much higher steady state concentrations of PE.l Synthesis of PE by reversal of the pyruvate kinase reaction was also obtained when respiring mitochondria were used as the ATP generator. Supplementation of this system with glycolytic enzymes led to formation of fructose-DP and other glycolytic intermediates.