Conformational effects of coenzyme binding to porcine lactic dehydrogenase.

Changes induced on addition of the coenzyme, NADH or NAD+, to porcine lactic dehydrogenase isoenzymes H4 and M4 have been studied by hydrodynamic and spectroscopic methods. As shown by ultracentrifugal analysis, the native subunit structure remains unchanged on holoenzyme formation; an approximately 5% increase of the sedimentation coefficient, parallelled by a slight decrease of the partial specific volume (less than 1%) indicate a significant change in the native tertiary and/or quaternary structure of the enzymes, corroborating earlier calorimetric data (Hinz and Jaenicke, 1975). The binding constant for the enzyme from skeletal muscle (M4) and NADH are found to be in agreement with KD-values obtained from equilibrium dialysis, as well as spectroscopic and thermal titration experiments (8 muM). Far UV circular dichroism measurements do not show significant changes on ligand binding, indicating unchanged helicity or compensatory conformational effects. In the near UV, ligand binding is reflected by an extrinsic Cotton effect around 340 nm; in the range of aromatic absorption no changes are detectable. The experimental results suggest that there are gross structural changes on coenzyme binding to lactic dehydrogenase which do not affect the intrinsic spectral properties normally applied to analyze transconformation reactions in protein molecules.