The resolution of some steps of the reactions of lactate dehydrogenase with its substrates |
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| Authors: | H D'A Heck C H McMurray and H Gutfreund |
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| Institution: | Max Planck Institut für Physikalische Chemie, Gottingen, Germany, and Department of Biochemistry, University of Bristol |
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| Abstract: | 1. The reaction of pig heart lactate dehydrogenase (EC 1.1.1.27) with NAD(+) and lactate to form pyruvate and NADH was followed by rapid spectrophotometric methods. The distinct spectrum of enzyme-bound NADH permits the measurement of the rate of dissociation of this compound. 2. The reduction of the first mole equivalent of NAD(+) per mole of enzyme sites can also be observed, and is much more rapid than the steady-state rate of NADH production. 3. At pH8 the dissociation of the enzyme-NADH complex is rate-determining for the steady-state oxidation of lactate. At lower pH some other step after the interconversion of the ternary complex and before the dissociation of NADH is rate-determining. Other evidence for a compulsory-order mechanism is provided. |
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