The nature of the substrate inhibition in lactate dehydrogenases as studied by a spin-labeled derivative of NAD+

  • Trommer W
  • Huth H
  • Wenzel H
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The formation of the ternary complex of lactate dehydrogenase (l-lactae:NAD+oxidoreductase, EC from pig heart and skeletal muscle with the adduct of pyruvate to NAD+, spin-labeled at N6was studied by ultraviolet spectroscopy and ESR techniques. According to ultraviolet measurements we found identical binding characteristics for the natural coenzyme and its spin-labeled analog. The rate by which the ESR signal of free spin-labeled NAD+decreased upon addition of pyruvate to the binary complexes was substantially different in the two isozymes. With the heart type an initial drop followed by a further linear decrease, zero order in the enzyme and coenzyme concentration was observed. In case of the skeletal muscle isozyme no immediate reaction and a first order process occurred. The initial reaction can be attributed to a non-covalent enzyme/spin-labeled NAD+/pyruvate complex with a dissociation constant for pyruvate of 11 ± 1 mM, thus explaining the well-known substrate inhibition in the heart isozyme above 2 mM pyruvate. The further reaction is then determined by the buffer dependent enolization of pyruvate. In the muscle isozyme formation of the covalent adduct is not assisted by prior binding of pyruvate in a non-covalent ternary complex and therefore the rate depends on the binary complex concentration. © 1979.

Author-supplied keywords

  • ESR
  • Lactate dehydrogenase
  • NAD+derivative
  • Pyruvate binding
  • Spin label

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  • Wolfgang E. Trommer

  • Helga Huth

  • Herbert R. Wenzel

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