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 o f the beta1beta1 isoenzyme of human alcohol dehydrogenase that were pr epared from dimeric enzyme by freeze-thaw in liquid nitrogen. Whereas the dimeric enzyme has optimal activity at low substrate concentration s (2.5 mM ethanol, 50 muM NAD+; ''low K(m)'' activity), the monomer ha s its highest activity at high substrate concentrations (1.5 M ethanol , 2.5 mM NAD+; ''high K(m)'' activity). While the activity of the mono mer does not appear to be saturated at 1.5 M ethanol, its maximal acti vity at this high ethanol concentration exceeds the V(max) of the dime r by about 3-fold. The apparent K(m) of NAD+ with monomers is 270 muM, and no activity could be detected with nicotinamide mononucleotide as cofactor. During gel filtration the high K(m) activity elutes at a lo wer apparent molecular weight position than the dimer. The kinetics of monomer-to-dimer reassociation are consistent with a second-order pro cess with a rate constant of 240 M-1 s-1. The reassociation rate is ma rkedly enhanced by the presence of NAD+. During refolding of beta1beta 1 following denaturation in 6 M guanidine hydrochloride, an enzyme spe cies with high K(m) activity and spectral properties similar to the fr eeze-thaw monomer is observed, indicating that a catalytically active monomer is an intermediate in the refolding pathway. The enzymatic act ivity of the monomers implies that the intersubunit contacts of beta1b eta1 are not crucial in establishing a catalytically competent enzyme. However, the differences in specific activity and K(m) between monome r and dimer suggest that dimerization may serve to modulate the cataly tic properties.