An ultraviolet resonance Raman study of dehydrogenase enzymes and their interactions with coenzymes and substrates.
Ultraviolet resonance Raman (UVRR) spectra, with 260-nm excitation, are reported for oxidized and reduced nicotinamide adenine dinucleotides (NAD+ and NADH, respectively). Corresponding spectra are reported for these coenzymes when bound to the enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and liver and yeast alcohol dehydrogenases (LADH and YADH). The observed differences between the coenzyme spectra are interpreted in terms of conformation, hydrogen bonding, and general environment polarity differences between bound and free coenzymes and between coenzymes bound to different enzymes. The possibility of adenine protonation is discussed. UVRR spectra with 220-nm excitation also are reported for holo- and apo-GAPDH (GAPDH-NAD+ and GAPDH alone, respectively). In contrast with the 260-nm spectra, these show only bands due to vibrations of aromatic amino acid residues of the protein. The binding of coenzyme to GAPDH has no significant effect on the aromatic amino acid bands observed. This result is discussed in the light of the known structural change of GAPDH on binding coenzyme. Finally, UVRR spectra with 240-nm excitation are reported for GAPDH and an enzyme-substrate intermediate of GAPDH. Perturbations are reported for tyrosine and tryptophan bands on forming the acyl enzyme.
Austin
J C
JC
Chemistry Department, University of York, Heslington, U.K.
Wharton
C W
CW
Hester
R E
RE
D000426:Alcohol Dehydrogenase / Q000378:metabolism*; D000818:Animals; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D006860:Hydrogen Bonding; D008099:Liver / Q000201:enzymology; D009132:Muscles / Q000201:enzymology; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013059:Spectrum Analysis, Raman / Q000379:methods
D009243:NAD; D000426:Alcohol Dehydrogenase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases