In vivo regulation of glycolysis and characterization of sugar: phosphotransferase systems in Streptococcus lactis

Abstract

Two novel procedures were used to regulate, in vivo, the formation of phosphoenolpyruvate (PEP) from glycolysis in S. lactis ML3. In the 1st procedure, glucose metabolism was specifically inhibited by p-chloromercuribenzoate. Autoradiographic and enzymatic analyses showed that the cells contained G-6-P, fructose 6-phosphate, fructose-1,6-diphosphate and triose phosphates. Dithiothreitol reversed the p-chloromercuribenzoate inhibition, and these intermediates were rapidly and quantitatively transformed into 3- and 2-phosphoglycerates plus PEP. The 3 intermediates were not further metabolized and constituted the intracellular PEP potential. The 2nd procedure simply involved starvation of the organisms. The starved cells were devoid of G-6-P, fructose 6-phosphate, fructose-1,6-diphosphate and triose phosphates but contained high levels of 3- and 2-phosphoglycerates and PEP (approximately 40 mM in total). The capacity to regulate PEP formation in vivo permitted the characterization of glucose and lactose phosphotransferase systems in physiologically intact cells. Evidence was obtained for feed forward activation of pyruvate kinase in vivo by phosphorylated intermediates formed before the glyceraldehyde-3-phosphate dehydrogenase reaction in the glycolytic sequence. Pyruvate kinase (an allosteric enzyme) probably plays a key role in the regulation of glycolysis and phosphotransferase system functions in S. lactis ML3.