Probing mechanisms of catalysis and electron transfer by methylamine dehydrogenase by site-directed mutagenesis of αPhe~(55)

摘要

Methylamine dehydrogenase (MADH) possesses an α_2β_2 subunit structure with each smaller β subunit possessing a tryptophan tryptophylquinone (TTQ) prosthetic group. Phe~(55) of the α subunit is located where the substrate channel from the enzyme surface opens into the active site. Site-directed mutagenesis studies have revealed several roles for this residue in catalysis and electron transfer (ET) by MADH. Site-directed mutagenesis of either αPhe~(55) or βIle~(107) (a residue in the β subunit which interacts with αPhe~(55)) converts MADH into enzymes with specificities for long-chain amines, amylamine or propylamine. Mutation of αPhe~(55) also affects monovalent cation binding to the active site. αF55A MADH exhibits an increased K_d for cation-dependent spectral changes and a decreased K_d for cation-dependent stimulation of the rate of gated ET from N-quinol MADH to amicyanin. These results demonstrate that αPhe~(55) is able to directly participate in a wide range of biochemical processes not typically observed for a phenylalanine residue.