ENZYME BASIS FOR PH REGULATION OF CITRATE AND PYRUVATE METABOLISM BY LEUCONOSTOC-OENOS

Citrate and pyruvate metabolism by nongrowing cells of Leuconostoc oen os was investigated. C-13 nuclear magnetic resonance (NMR) spectroscop y was used to elucidate the pathway of citrate breakdown and to probe citrate or pyruvate utilization, noninvasively, in living cell suspens ions. The utilization of isotopically enriched substrates allowed us t o account for the end products derived from the metabolism of endogeno us reserves. The effect of environmental parameters, e.g., pH, gas atm osphere, and presence of malate, on the end products of citrate utiliz ation was studied. Approximately 10% of the citrate supplied was conve rted to aspartate which remained inside the cells. A metabolic shift w ith pH was observed, with acetoin production being favored at pH 4, wh ereas lactate and acetate production increased significantly at higher pH values. The information obtained with NMR was complemented with st udies on the relevant enzyme activities in the metabolic pathway of ci trate breakdown. The intracellular pH of the cells was strongly depend ent on the external pH; this result, together with the determination o f the pH profile of the enzymic activities, allowed us to establish th e basis for pH regulation; lactate dehydrogenase activity was optimal at pH 7, whereas the acetoin-forming enzymes displayed maximal activit ies below pH 5. Citrate utilization was also monitored in dilute cell suspensions for comparison with NMR experiments performed with dense s uspensions.