Johannes Everse

Addition products of diphosphopyridine nucleotides with substrates of pyridine nucleotide-linked dehydrogenases☆

Abstract Preparation of the reduced and oxidized forms of addition compounds from diphosphopyridine nucleotide and a number of carbonyl compounds are described; these include pyruvate, pyruvic ethylester, oxaloacetate, α-ketoglutarate, acetaldehyde, butyraldehyde, and α-ketobutyrate. The adducts are closely related to the DPN-acetone and the DPN-dihydroxyacetone adducts, both in their chemical and spectral properties. The chemical structures of the reduced and oxidized adducts have been determined and information concerning the mechanism of formation of the adducts is presented. The lactate, malate, glutamate, and alcohol dehydrogenases show a unique specificity for the reduced adducts; the specificity was found to be related to the normal oxidized substrate utilized by the various enzymes. The inhibition is competitive with DPNH and noncompetitive with the substrate. Oxidized adducts produce considerably less inhibitory effects than the reduced adducts. Evidence is presented showing that the reduced adducts bind to the enzymes in a manner similar to that of the reduced coenzyme. On the basis of the high selectivity of the dehydrogenases for the adducts, a model is proposed which shows the spatial relationship between the reduced coenzyme and the oxidized substrate on the enzyme in the transition state. From this model, a general reaction mechanism is postulated for the pyridine nucleotide-dependent dehydrogenases, which may explain the stereo-specificity of the hydrogen transfer with respect to the coenzyme as well as the substrate.

The formation of ternary complexes by diphosphopyridine nucleotide-dependent dehydrogenases

Abstract Formation of abortive ternary complexes between enzyme, coenzyme, and substrate has been demonstrated for several pyridine nucleotide-dependent dehydrogenases by measuring the quenching of protein fluorescence. Evidence is presented suggesting the participation of a ternary complex consisting of lactate dehydrogenase, DPN + , and pyruvate in the phenomenon of substrate inhibition observed with lactate dehydrogenases. This complex is more readily formed with the H-type LDH than with the M-type enzyme under identical conditions. The ternary complex of LDH-DPN + -pyruvate can be formed at physiological levels of pyruvate. Information concerning the structure of the ternary complex has been derived from its absorption and fluorescence spectra, as well as from its kinetic and chemical properties. The pyridine ring in the complex appears to be in the reduced form, similar to that found in DPNH. The ternary complex can be precipitated with ammonium sulfate. Denaturation of the enzyme results in a product which is easily oxidized. Evidence is presented indicating that this product consists of DPN which is covalently bonded to pyruvate. Similar products can be obtained with analogs of DPN, but the reaction for lactate dehydrogenase is specific for pyruvate. Related ternary complexes have been observed from malate dehydrogenase, glutamic dehydrogenase, and alcohol dehydrogenase. The significance of ternary complex formation is discussed in relation to the reaction mechanism of pyridine nucleotide-linked dehydrogenases.

Accumulation of oxidized diphosphopyridine nucleotide in mouse liver after administration of reduced diphosphopyridine nucleotide

Abstract The intraperitoneal injection of large amounts of pyridine nucleotide coenzymes into mice results in an elevated level of liver pyridine nucleotides. These pyridine nucleotides are mainly present in the oxidized form. The highest levels of liver coenzymes are observed after the administration of DPNH. Administration of the coenzymes per os also resulted in an increase in liver DPN + . Studies with radioactive coenzymes indicate that the pyridine nucleotides may enter the liver without cleavage.