Title
Studying NAD(P)H cofactor-binding to alcohol dehydrogenases through global analysis of circular dichroism spectra
Author
Marija Marolt, Steffen Lüdeke
Year
2018
Journal
Physical Chemistry Chemical Physics
Abstract
The initial step in reactions catalyzed by NAD(P)H-dependent alcohol dehydrogenases (ADHs) is the binding of the cofactor to the active site. On the basis of a law of mass action-model, we determined the proportions of binding and non-binding NAD(P)H and the associated dissociation constants (Kd) from matrix-least squares global fitting of the NAD(P)H concentration-dependent circular dichroism (CD) spectra in presence of purified enzymes (ADH from horse liver, HLADH; ADH-A from Rhodococcus ruber; YGL157w from Saccharomyces cerevisiae) or enzyme-containing whole cell extract (ADH from Lactobacillus brevis, LbADH). Furthermore, the fitting allowed the back calculation of CD spectra corresponding to the cofactor in its bound conformation. At increasing pH and/or increasing ionic strength we detected an increase of Kd for the NADH HLADH complex with the shape of the bound cofactor conformation spectrum remaining unaffected. While the bound cofactor spectrum for the ADH-A·NADH complex was similar as for HLADH, the corresponding spectra obtained for the NADPH-dependent enzymes YGL157w and LbADH exhibited spectra with opposite sign of the most prominent band. By comparison to CD spectra calculated on cofactor geometries from crystal structures at the sTD-DFT level, we found that the sign of the bound cofactor spectrum correlates with the orientation of the nicotinamide ring of the cofactor in the active site. These results demonstrate the usefulness of a global analysis of cofactor titration CD spectra to study the role of cofactor binding and its geometry in ADH catalysis.
Instrument
J-810
Keywords
Circular dichroism, Protein structure, Ligand binding, Chemical stability, Biochemistry