Deuterium isotope effects on lactate dehydrogenase using L-2-hydroxysuccinamate and effect of an inhibitor in the variable substrate on observed isotope effects.
L-2-Hydroxysuccinamate, the 4-amide analogue of L-malate, is a substrate for beef heart lactate dehydrogenase with values for V and V/K at pH 8 which are 0.01 and 0.0002% of the corresponding constants for L-lactate and an equilibrium constant of 2.19 X 10(-13) M. Similar values are observed for rabbit muscle lactate dehydrogenase. With beef heart lactate dehydrogenase, the pH-independent isotope effects on V and V/K of 3.7 +/- 0.5 and 3.3 +/- 0.4 indicate that hydride transfer is largely rate limiting for this reaction. L-2-Hydroxysuccinamate undergoes hydrolysis in mildly acidic or basic solution, and the pH vs. rate profile suggests intramolecular catalysis by the undissociated 1-carboxyl group in the pH range 1.5--3.5. The substrate activity of L-2-hydroxysuccinamate with pigeon liver malic enzyme reported by M. I. Schimerlik & W. W. Cleland [(1977) Biochemistry 16, 565] was caused by contaminating L-malate; the purified compound shows no activity (<0.015%). Theory has been developed for the effect on V and V/K deuterium isotope effects of having an inhibitor present in the variable substrate and tested by adding trifluoroethanol to deuterated or unlabeled cyclohexanol as substrates for alcohol dehydrogenase.
D000577:Amides / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D003903:Deuterium; D007553:Isotope Labeling; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D008433:Mathematics; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D009904:Optical Rotation; D011817:Rabbits; D013386:Succinates / Q000494:pharmacology*
D000577:Amides; D013386:Succinates; C016511:2-hydroxysuccinamate; D003903:Deuterium; D007770:L-Lactate Dehydrogenase