Monomers of human beta 1 beta 1 alcohol dehydrogenase exhibit activity that differs from the dimer.
A previously unreported enzymatic activity is described for monomers of the beta 1 beta 1 isoenzyme of human alcohol dehydrogenase that were prepared from dimeric enzyme by freeze-thaw in liquid nitrogen. Whereas the dimeric enzyme has optimal activity at low substrate concentrations (2.5 mM ethanol, 50 microM NAD+; "low Km" activity), the monomer has its highest activity at high substrate concentrations (1.5 M ethanol, 2.5 mM NAD+; "high Km" activity). While the activity of the monomer does not appear to be saturated at 1.5 M ethanol, its maximal activity at this high ethanol concentration exceeds the Vmax of the dimer by about 3-fold. The apparent Km of NAD+ with monomers is 270 microM, and no activity could be detected with nicotinamide mononucleotide as cofactor. During gel filtration the high Km activity elutes at a lower apparent molecular weight position than the dimer. The kinetics of monomer-to-dimer reassociation are consistent with a second-order process with a rate constant of 240 M-1 s-1. The reassociation rate is markedly enhanced by the presence of NAD+. During refolding of beta 1 beta 1 following denaturation in 6 M guanidine hydrochloride, an enzyme species with high Km activity and spectral properties similar to the freeze-thaw monomer is observed, indicating that a catalytically active monomer is an intermediate in the refolding pathway. The enzymatic activity of the monomer implies that the intersubunit contacts of beta 1 beta 1 are not crucial in establishing a catalytically competent enzyme. However, the differences in specific activity and Km between monomer and dimer suggest that dimerization may serve to modulate the catalytic properties.
Ehrig
T
T
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122.
Muhoberac
B B
BB
Brems
D
D
Bosron
W F
WF
D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D009243:NAD / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D006146:Guanidines; D007527:Isoenzymes; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D009243:NAD; D000426:Alcohol Dehydrogenase; D019791:Guanidine