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 dehydrogenase (MADH) with the physiological substrates methylamine and amicyanin show a pH profile that is quite different from the one found in commonly used assays with artificial electron acceptors. The optimum at pH 7.5, observed for kcat in the latter case, is absent with amicyanin as the reoxidizing substrate. With amicyanin kcat scarcely depends on pH; the same is true for the maximal rate of reduction of MADH by methylamine (kred). Conversely, both the specificity constant (kcat/Km) of MADH for amicyanin and the apparent second-order rate constant for the reduction of MADH by methylamine (kassocapp) 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, which only binds the larger cations (Cs+, Rb+, and NH4+), is accompanied by a red shift in the absorbance spectrum, whereas cation binding to the low-affinity site, which, less specifically, favors binding of the smaller cations, leads to a bleaching of the visible spectrum with a concomitant increase in the near-UV. Cation binding to either site strongly affects the reactivity of MADH. The reduction of MADH by methylamine is inhibited by monovalent cations, whereas the oxidation of reduced MADH by amicyanin is strongly stimulated. For the former reaction it was established that cations affect only kassocapp, not kred. Some speculations about the molecular basis for the effects of pH and cations are presented.
Gorren
A C
AC
Department of Microbiology and Enzymology, Delft University of Technology, The Netherlands.
Duine
J A
JA
D001426:Bacterial Proteins / Q000378:metabolism; D002414:Cations, Monovalent; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008744:Methylamines / Q000378:metabolism; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry* / Q000378:metabolism; D013055:Spectrophotometry, Infrared; D013056:Spectrophotometry, Ultraviolet; D013855:Thiobacillus / Q000201:enzymology*
D001426:Bacterial Proteins; D002414:Cations, Monovalent; D008744:Methylamines; C017079:mauC protein, Methylobacterium extorquens; C027451:methylamine; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors