Abstract
A study of difference spectra in the range, 300–500 mμ is reported involving highly purified rat liver lactate dehydrogenase (LDH) and oxidized or reduced coenzyme or coenzyme analog (nicotinamide-adenine dinucleotide (NAD) or acetylpyridine-adenine dinucleotide (AcPyAD). The formation of the binary complexes, LDH · NAD or LDH · AcPyAD is associated with the development of spectral bands with λ max at 335 mμ and 355 mμ, respectively. The observed absorbance is proportional to coenzyme concentration at fixed LDH concentration. The spectral behavior of the LDH · oxidized coenzyme binary complexes is clearly distinguishable from that of the binary complexes involving NADH or AcPyADH. A conclusion is that the binding of oxidized coenzymes to LDH results in a significant change in the chromophoric behavior of a hybridized form of the substituted pyridine ring. The formation of abortive ternary complexes involving LDH · oxidized coenzyme /sd pyruvate or LDH · oxidized coenzyme · /ga-ketobutyrate was accompanied by slow progressive spectral changes which reached equilibrium values, dependent on the concentration of all three reactants. For LDH · NAD · pyruvate λ max, was 323 mμ; for LDH · AcPyAD · pyruvate λ max was 341 mμ. The spectral changes were fully reversible upon dialysis vs. AcPyAD-buffer in the one case but not upon dialysis vs. NAD-buffer in the other. Complete recovery of enzyme activity was noted in each case after dialysis to remove pyruvate or α-ketobutyrate. The spectral changes are considered to be those associated with the allosteric binding of pyruvate or α-ketobutyrate to binary complexes of LDH · oxidized coenzyme with resulting conformational changes in LDH which affect the active sites. No abortive ternary complex involving LDH-reduced coenzyme (or reduced coenzyme analog) -lactate could be detected by spectral means.
Published Version
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