EVIDENCE FOR A METHYLAMMONIUM-BINDING SITE ON METHYLAMINE DEHYDROGENASE OF THIOBACILLUS-VERSUTUS

The nonconvertible substrate analogues di-, tri-, and tetramethylammonium are bound with fairly high affinity to oxidized methylamine dehydrogenase (MADH(ox)) from Thiobacillus versutus and induce the same red-shift in the optical absorbance spectrum of MADH(ox) as do the monovalent cations Cs+, Rb+, and NH4+. Like the monovalent cations, trimethylamine also competitively inhibits the reduction of MADH(ox) by methylamine. Rapid-scan experiments show that within the first few milliseconds of the reaction between MADH(ox) and methylamine a fed-shifted intermediate is formed as well. Taken together these experiments demonstrate the existence of a common binding site on MADH(ox) for the substrate CH3NH3+, the substrate analogues (CH3)(2)NH2+, (CH3)(3)NH+ and (CH3N+, and the monovalent cations Cs+, Rb+, and N-4(+). Therefore we conclude that, prior to conversion, methylamine is noncovalently bound to MADH(ox), as a cation. The resonance Raman spectra of MADH(ox) in the absence and presence of Cs+, NH4+, and (CH3)(3)NH+ are very similar, except for the C=O stretching frequencies of the o-quinone carbonyls of the tryptophyltryptophanquinone (TTQ) active center, which show 5-30 cm(-1) downshifts. From these Raman results and the X-ray crystal structure, we conclude that the CH3NH3+ binding site is in close proximity to the 06 carbonyl oxygen of the TTQ.