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

The nonconvertible substrate analogues di-, tri-, and tetramethylammon ium are bound with fairly high affinity to oxidized methylamine dehydr ogenase (MADH(ox)) from Thiobacillus versutus and induce the same red- shift in the optical absorbance spectrum of MADH(ox) as do the monoval ent cations Cs+, Rb+, and NH4+. Like the monovalent cations, trimethyl amine also competitively inhibits the reduction of MADH(ox) by methyla mine. Rapid-scan experiments show that within the first few millisecon ds of the reaction between MADH(ox) and methylamine a fed-shifted inte rmediate is formed as well. Taken together these experiments demonstra te the existence of a common binding site on MADH(ox) for the substrat e CH3NH3+, the substrate analogues (CH3)(2)NH2+, (CH3)(3)NH+ and (CH3N +, and the monovalent cations Cs+, Rb+, and N-4(+). Therefore we concl ude that, prior to conversion, methylamine is noncovalently bound to M ADH(ox), as a cation. The resonance Raman spectra of MADH(ox) in the a bsence and presence of Cs+, NH4+, and (CH3)(3)NH+ are very similar, ex cept for the C=O stretching frequencies of the o-quinone carbonyls of the tryptophyltryptophanquinone (TTQ) active center, which show 5-30 c m(-1) downshifts. From these Raman results and the X-ray crystal struc ture, we conclude that the CH3NH3+ binding site is in close proximity to the 06 carbonyl oxygen of the TTQ.