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.
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122.
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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