FEMS Microbiology Letters 22 (1984) 73-76 73 Published by Elsevier Regulation of lactate dehydrogenase in Brochothrix thermosphacta (Lactate dehydrogenase; 31p_NMR, Brochothrix thermosphacta; homolactic fermentation; fructose-l,6-bisphosphate; regulation) Satya P. Singh, Robert Vink and Peter J. Rogers Schoolof Science, Griffith University,Brisbane4111, Australia Received24 October 1983 Revision receivedand accepted5 December1983 1. SUMMARY Purified lactate dehydrogenase from Brochothrix thermosphacta is stimulated by Fru-l,6-P 2 and G6P although saturating concentrations are high (> 20 mM). Neither is essential for activity. AMP, ADP and ATP inhibit enzyme activity consistent with either non-competitive (with Fru-l,6-P 2 present) or uncompetitive (G6P present) inhibition. Activity is not dependent on Pi (< 200 mM). Based on 31p_ NMR of cells, sugar phosphate concentration can reach 30 mM with excess glucose present; NDP and NTP also accumulate to levels that inhibit the isolated enzyme. The effector levels in vitro are therefore appropriate to in vivo metabolism and support a regulatory role for sugar phosphates during pyruvate metabolism in this organism. 2. INTRODUCTION Brochothrix thermosphacta (formerly Micro- bacterium thermosphactum) is a facultative anaerobe that is related to the Streptococci [1]. Abbreviations: Fru-l,6-P 2, fructose-l,6-bisphosphate; G6P, glucose-6-phosphate; LDH, lactate dehydrogenase; Pi, in- organic phosphate. Anoxic cultures produce lactic acid when glucose is in excess (> 1 mM); acetate, ethanol and for- mate are favoured under glucose-limited, steady state conditions [2,3], apparently because pyru- vate-formate-lyase, the anaerobic counterpart of pyruvate dehydrogenase, competes with LDH [2]. In Streptococcus mutans, the purified lyase, is in- hibited by triose phosphates [4] (<0.5 mM) whereas LDH activity in some Streptococci is stimulated by Fru-l,6-P 2 (review [5]). Pyruvate metabolism may therefore reflect the concentra- tion of glycolytic intermediates and perhaps p. 4, 5 since it acts as a general inhibitor of Fru-l,6-PE-de- pendent LDHs [5]. We have purified LDH and related the in vitro properties to the kinetics of glucose metabolism in intact cells in B. thermo- sphacta using 31p-NMR. The enzyme is unique in that both Fru-l,6-P 2 and G6P are activators, al- though high concentrations are required for saturation. ATP and ADP are inhibitory whilst Pi has no significant effect. 31p-NMR of intact cells shows that cytoplasmic NDP and NTP can be within the inhibitory range and that sugar phos- phates accumulate to at least 30 mM with excess glucose present. The in vitro effector levels are therefore appropriate to physiological conditions, and indicate that LDH activity and the pathway for pyruvate metabolism are dependent on sugar phosphate concentration. 0378-1097/84/$03.00 © 1984 Federation of European Microbiological Societies