Phosphoenolpyruvate and 2-phosphoglycerate: endogenous energy source(s) for sugar accumulation by starved cells of Streptococcus lactis

Abstract

In the absence of an exogenous energy source, galactose-grown cells of S. lactis ML3 rapidly accumulated thiomethyl-.beta.-D-galactopyranoside (TMG) and 2-deoxyglucose to intracellular concentrations of 40-50 mM. Starved cells maintained the capacity for TMG uptake for many hours, and accumulation of the .beta.-galactoside was insensitive to proton-conducting ionophores (tetrachlorosalicylanilide and carbonylcyanide-m-chlorophenyl hydrazone) and SH group reagents including iodoacetate and N-ethylmaleimide. Fluorimetric analysis of glycolytic intermediates in extracts prepared from starved cells revealed (a) high intracellular levels of phosphoenolpyruvate (13 mM; PEP) and 2-phosphoglycerate (.apprx. 39 mM; 2-PG), but (b) an absence of other metabolites including G-6-P, fructose 6-phosphate, fructose 1,6-diphosphate, and triosephosphates. The following criteria showed PEP (and 2-PG) to be the endogenous energy source for TMG accumulation by the phosphotransferase system: the intracellular concentrations of PEP and 2-PG decreased with concomitant uptake of TMG, and a close correlation was observed between maximum accumulation of the .beta.-galactoside and the total available concentration of the 2 intermediates; TMG accumulated as an anionic derivative, which after extraction and incubation with alkaline phosphatase (EC 3.1.3.1) formed the original analog; fluoride inhibition of 2-phospho-D-glycerate hydrolyase (EC 4.2.1.11) prevented the conversion of 2-PG to PEP, and uptake of TMG by the starved cells was reduced by 80%; and the stoichiometric ratio [TMG] accumulated/[PEP] consumed was almost unity (0.93). In cells metabolizing glucose, all intermediates listed in (a) and (b) were found. Upon exhaustion of glucose from the medium, the metabolites in (b) were not longer detectable, while the intracellular concentrations of PEP and 2-PG increased to the levels previously observed in starved cells. The glycolytic intermediates in (b) are all in vitro heterotropic effectors of pyruvate kinase ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from S. lactis ML3. The capacity of starved cells to maintain high intracellular concentrations of PEP and 2-PG is probably a consequence of decreased in vivo activity of this key regulatory enzyme of glycolysis.