Enzyme Basis for pH Regulation of Citrate and Pyruvate Metabolism by Leuconostoc oenos

Citrate and pyruvate metabolism by nongrowing cells of Leuconostoc oenos was investigated. 13C nuclear magnetic resonance (NMR) spectroscopy was used to elucidate the pathway of citrate breakdown and to probe citrate or pyruvate utilization, noninvasively, in living cell suspensions. The utilization of isotopically enriched substrates allowed us to account for the end products derived from the metabolism of endogenous reserves. The effect of environmental parameters, e.g., pH, gas atmosphere, and presence of malate, on the end products of citrate utilization was studied. Approximately 10% of the citrate supplied was converted to aspartate which remained inside the cells. A metabolic shift with pH was observed, with acetoin production being favored at pH 4, whereas lactate and acetate production increased significantly at higher pH values. The information obtained with NMR was complemented with studies on the relevant enzyme activities in the metabolic pathway of citrate breakdown. The intracellular pH of the cells was strongly dependent on the external pH; this result, together with the determination of the pH profile of the enzymic activities, allowed us to establish the basis for pH regulation; lactate dehydrogenase activity was optimal at pH 7, whereas the acetoin-forming enzymes displayed maximal activities below pH 5. Citrate utilization was also monitored in dilute cell suspensions for comparison with NMR experiments performed with dense suspensions.