Kinetic and structural studies on the catalytic role of the aspartic acid residue conserved in copper amine oxidase.
Copper amine oxidase contains a post-translationally generated quinone cofactor, topa quinone (TPQ), which mediates electron transfer from the amine substrate to molecular oxygen. The overall catalytic reaction is divided into the former reductive and the latter oxidative half-reactions based on the redox state of TPQ. In the reductive half-reaction, substrate amine reacts with the C5 carbonyl group of the oxidized TPQ, forming the substrate Schiff base (TPQ(ssb)), which is then converted to the product Schiff base (TPQ(psb)). During this step, an invariant Asp residue with an elevated pKa is presumed to serve as a general base accepting the alpha proton of the substrate. When Asp298, the putative active-site base in the recombinant enzyme from Arthrobacter globiformis, was mutated into Ala, the catalytic efficiency dropped to a level of about 10(6) orders of magnitude smaller than the wild-type (WT) enzyme, consistent with the essentiality of Asp298. Global analysis of the slow UV/vis spectral changes observed during the reductive half-reaction of the D298A mutant with 2-phenylethylamine provided apparent rate constants for the formation and decay of TPQ(ssb) (k(obs) = 4.7 and 4.8 x 10(-4) s(-1), respectively), both of which are markedly smaller than those of the WT enzyme determined by rapid-scan stopped-flow analysis (k(obs) = 699 and 411 s(-1), respectively). Thus, Asp298 plays important roles not only in the alpha-proton abstraction from TPQ(ssb) but also in other steps in the reductive half-reaction. X-ray diffraction analyses of D298A crystals soaked with the substrate for 1 h and 1 week revealed the structures of TPQ(ssb) and TPQ(psb), respectively, as pre-assigned by single-crystal microspectrophotometry. Consistent with the stereospecificity of alpha-proton abstraction, the pro-S alpha-proton of TPQ(ssb) to be abstracted is positioned nearly perpendicularly to the plane formed by the Schiff-base imine double bond conjugating with the quinone ring of TPQ, so that the orbitals of sigma and pi electrons maximally overlap in the conjugate system. More intriguingly, the pro-S alpha proton of the substrate is released stereospecifically even in the reaction catalyzed by the base-lacking D298A mutant. On the basis of these results, we propose that the stereospecificity of alpha-proton abstraction is primarily determined by the conformation of TPQ(ssb), rather than the relative geometry of TPQ and the catalytic base.
Chiu
Yen-Chen
YC
Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.
Okajima
Toshihide
T
Murakawa
Takeshi
T
Uchida
Mayumi
M
Taki
Masayasu
M
Hirota
Shun
S
Kim
Misa
M
Yamaguchi
Hiroshi
H
Kawano
Yoshiaki
Y
Kamiya
Nobuo
N
Kuroda
Shun'ichi
S
Hayashi
Hideyuki
H
Yamamoto
Yukio
Y
Tanizawa
Katsuyuki
K
D006631:Amine Oxidase (Copper-Containing) / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology; D001224:Aspartic Acid / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D012545:Schiff Bases / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman
D012545:Schiff Bases; D001224:Aspartic Acid; D004295:Dihydroxyphenylalanine; C067626:6-hydroxydopa quinone; D006631:Amine Oxidase (Copper-Containing)