AROMATIC AMINE DEHYDROGENASE, A 2ND TRYPTOPHAN TRYPTOPHYLQUINONE ENZYME

Aromatic amine dehydrogenase (AADH) catalyzes the oxidative deaminatio n of aromatic amines including tyramine and dopamine. AADH is structur ally similar to methylamine dehydrogenase (MADH) and possesses the sam e tryptophan tryptophylquinone (TTQ) prosthetic group. AADH exhibits a n alpha(2) beta(2) structure with subunit molecular weights of 39,000 and 18,000 and with a quinone covalently attached to each beta subunit . Neither subunit cross-reacted immunologically with antibodies to the corresponding subunits of MADH, and the N-terminal amino acid sequenc e of the beta subunit of AADH exhibited no homology with the highly co nserved beta subunits of MADH. The absorption spectra for the oxidized , semiquinone, and reduced forms of AADH have been characterized, and extinction coefficients for the absorption maxima of each redox form h ave been determined. These spectra are very similar to those for MADH, indicating the likelihood of a TTQ cofactor. This was verified by the near identity of the vibrational frequencies and intensities in the r esonance Raman spectra for the oxidized forms of AADH and MADH. A stab le semiquinone of AADH could be observed during a reductive titration with dithionite, whereas titration with tyramine proceeded directly fr om the oxidized to the reduced form. AADH was very stable against dena turation by heat and exposure to guanidine. The individual subunits co uld be separated by gel filtration after incubation in guanidine hydro chloride, and partial reconstitution of activity was observed on recom bination of the subunits. Steady-state kinetic analysis of AADH yielde d a V-max of 17 mu mol/min/mg and K-m for tyramine of 5.4 mu M. Substr ate inhibition by tyramine was observed. AADH was irreversibly inhibit ed by hydrazine, phenylhydrazine, hydroxylamine, semicarbazide, and am inoguanidine. Isonicotinic acid hydrazide (isoniazid) and isonicotinic acid 2-isopropyl hydrazide (iproniazid) were reversible noncompetitiv e inhibitors of AADH and exhibited K-i values of 8 and 186 mu M, respe ctively. The similarities and differences between AADH and other amine oxidizing enzymes are also discussed.