The nonconvertible substrate analogues di-, tri-, and tetramethylammonium are bound with fairly high affinity to oxidized methylamine dehydrogenase (MADHOX) from Thiobacillus versutus and induce the same red-shift in the optical absorbance spectrum of MADHOX as do the monovalent cations Cs+, Rb+, and NH4+. Like the monovalent cations, trimethylamine also competitively inhibits the reduction of MADHox by methylamine. Rapid-scan experiments show that within the first few milliseconds of the reaction between MADHOX and methylamine a red-shifted intermediate is formed as well. Taken together these experiments demonstrate the existence of a common binding site on MADHOX for the substrate CH3NH3+ the substrate analogues (CH3NH2+, (CH3)3NH+, and (CH3)4N+, and the monovalent cations Cs+, Rb+, and NH4+. Therefore we conclude that, prior to conversion, methylamine is noncovalently bound to MADHox as a cation. The resonance Raman spectra of MADHOX in the absence and presence of Cs+, NH4+, and (CH3)3NH+ are very similar, except for the C=0 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 O6 carbonyl oxygen of the TTQ.
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