THE EFFECTS OF PH AND CATIONS ON THE SPECTRAL AND KINETIC-PROPERTIES OF METHYLAMINE DEHYDROGENASE FROM THIOBACILLUS-VERSUTUS

The catalytic parameters of Thiobacillus versutus methylamine dehydrog enase (MADH) with the physiological substrates methylamine and amicyan in show a pH profile that is quite different from the one found in com monly used assays with artificial electron accepters. The optimum at p H 7.5, observed for k(cat) in the latter case, is absent with amicyani n as the reoxidizing substrate. With amicyanin k(cat) scarcely depends on pH; the same is true for the, maximal rate of reduction of MADH by methylamine (k(red)). Conversely, both the specificity constant (k(ca t)/K-m) of MADH for amicyanin and the apparent second-order rate const ant for the reduction of MADH by methylamine (k(assoc)(app)) increase very sharply with pH. MADH has a high- and a low-affinity binding site for monovalent cations. Cation binding to the high-affinity site, whi ch only binds the larger cations (Cs+, Rb+, and NH4+), is accompanied by a red shift in the absorbance spectrum, whereas cation binding to t he low-affinity site, which, less specifically, favors binding of the smaller cations, leads td a bleaching of the visible spectrum with a c oncomitant increase in the near-UV. Cation binding to either site stro ngly affects the reactivity of MADH; The reduction of MADH by methylam ine is inhibited by monovalent cations, whereas the oxidation of reduc ed MADH by amicyanin is strongly stimulated. For the former reaction i t was established that cations affect only k(assoc)(app), not k(red). Some speculations about the molecular basis for the effects of pH and cations are presented.