The silent form of quinohemoprotein amine dehydrogenase from Paracoccus denitrificans.
Quinohemoprotein amine dehydrogenase (QH-AmDH) from Paracoccus denitrificans is an alphabetagamma heterotrimeric enzyme catalyzing oxidative deamination of amines with large substitutes, such as butylamine and benzylamine. The smallest gamma subunit has cross-linking cysteine tryptophylquinone (CTQ) as a catalytic center. A hemoprotein similar to QH-AmDH in molecular mass, subunit structure, and UV-vis spectral property, but having no enzymatic activity, was isolated. The enzymatically silent form (sQH-AmDH) was activated slowly by the substrates of QH-AmDH, and quickly by reductants, dithionite and dithiothreitol. Electrolysis of sQH-AmDH yielded the active form at potentials more negative than -0.17 V (vs. SHE). The activated protein reacted with a carbonyl reagent, 4-nitrophenylhydrazine, giving a typical spectrum of 4-nitrophenylhydrazone, while sQH-AmDH did not give such a spectrum. The gamma subunit of sQH-AmDH showed a sharp peak at 390 nm in UV-vis spectrum clearly distinguishable from that of QH-AmDH. Electrospray ionization mass spectrometric analysis showed that the molecular mass of the gamma subunit of sQH-AmDH was larger than that of QH-AmDH by 15.6. The data suggest that the CTQ-like moiety of sQH-AmDH is an oxime. This hypothesis was confirmed by subsequent hydroxylamine treatment of QH-AmDH. QH-AmDH was treated with hydroxylamine yielding an oxime derivative. The UV-vis spectral properties of the gamma subunit of hydroxylamine-treated QH-AmDH were identical to those of sQH-AmDH. The hydroxylamine-treated QH-AmDH was also reactivated by butylamine, as in the case of sQH-AmDH.
Fujieda
Nobutaka
N
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan. n-fujieda@iae.kyoto-u.ac.jp
Mori
Megumi
M
Ikeda
Tokuji
T
Kano
Kenji
K
D000042:Absorption; D000588:Amines / Q000494:pharmacology; D004572:Electrolysis; D004789:Enzyme Activation / Q000187:drug effects; D006420:Hemeproteins / Q000737:chemistry / Q000378:metabolism; D006835:Hydrazones / Q000737:chemistry / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry* / Q000378:metabolism*; D010231:Paracoccus denitrificans / Q000201:enzymology*; D019163:Reducing Agents / Q000494:pharmacology; D013056:Spectrophotometry, Ultraviolet
D000588:Amines; D006420:Hemeproteins; D006835:Hydrazones; D019163:Reducing Agents; D000587:Oxidoreductases Acting on CH-NH Group Donors; C119497:quinohemoprotein amine dehydrogenase