Classical Raman spectroscopic studies of NADH and NAD+ bound to liver alcohol dehydrogenase by difference techniques.
We report the Raman spectra of reduced and oxidized nicotinamide adenine dinucleotide (NADH and NAD+, respectively) and adenosine 5'-diphosphate ribose (ADPR) when bound to the coenzyme site of liver alcohol dehydrogenase (LADH). The bound NADH spectrum is calculated by taking the classical Raman difference spectrum of the binary complex, LADH/NADH, with that of LADH. We have investigated how the bound NADH spectrum is affected when the ternary complexes with inhibitors are formed with dimethyl sulfoxide (Me2SO) or isobutyramide (IBA), i.e., LADH/NADH/Me2SO or LADH/NADH/IBA. Similarly, the difference spectra of LADH/NAD+/pyrazole or LADH/ADPR with LADH are calculated. The magnitude of these difference spectra is on the order of a few percent of the protein Raman spectrum. We report and discuss the experimental configuration and control procedures we use in reliably calculating such small difference signals. These sensitive difference techniques could be applied to a large number of problems where the classical Raman spectrum of a "small" molecule, like adenine, bound to the active site of a protein is of interest. The spectrum of bound ADPR allows an assignment of the bands of the bound NADH and NAD+ spectra to normal coordinates located primarily on either the nicotinamide or the adenine moiety. By comparing the spectra of the bound coenzymes with model compound data and through the use of deuterated compounds, we confirm and characterize how the adenine moiety is involved in coenzyme binding and discuss the validity of the suggestion that the adenine ring is protonated upon binding. The nicotinamide moiety of NADH shows significant molecular changes upon binding.(ABSTRACT TRUNCATED AT 250 WORDS)
Chen
D
D
Physics Department, City College of The City University of New York, New York 10031.
Yue
K T
KT
Martin
C
C
Rhee
K W
KW
Sloan
D
D
Callender
R
R
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D003903:Deuterium; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D013059:Spectrum Analysis, Raman / Q000379:methods
D009243:NAD; D000246:Adenosine Diphosphate Ribose; D003903:Deuterium; D000426:Alcohol Dehydrogenase