PLANT LACTATE-DEHYDROGENASE - NADH KINETICS AND INHIBITION BY ATP

Lactate dehydrogenase (LDH), isolated from either turnip (Brassica rap a, Cruciferae) or from leek (Allium porrum, Liliaceae) shows normal Mi chaelis-Menten kinetics with NAD(+) in the lactate dehydrogenase direc tion, but non-hyperbolic kinetics with NADH in the pyruvate reductase direction. These kinetics are not 'sigmoidal' and appear consistently as two intersecting straight lines in reciprocal plots, representing a sharp decline in the effectiveness of NADH as a substrate in its lowe r concentration range. The overall apparent affinity for NADH decrease s with decreasing pH. Competitive inhibition by ATP, which is much str onger than that characteristic of mammalian LDH, also displays kinetic s that seem to be biphasic, but the K-i value does not vary significan tly with pH in the physiological range. The counter-inhibitory effect of Mg2+ ions is shown to be due to the formation of non-inhibitory Mg- ATP complexes. The dissociation of this is strongly pH dependent and t his results in most of the ATP being complexed, and therefore non-inhi bitory at pH 7.4 (the normal pH in plant cytosol) but largely uncomple xed and inhibitory at pH 6.4 (probably a lower cytosolic pH limit). Th ese factors, and particularly the last, combine to inhibit the enzyme more strongly as the pH drops (as a result of lactate-terminating anae robic glycolysis) and may act as an effective negative feed-back mecha nism in plant cells, allowing a pH-activated switchover to ethanol-ter minating glycolysis while preventing 'overshoot' into harmful acidosis . (C) 1997 Elsevier Science Ltd. All rights reserved.