Quantum mechanical hydrogen tunneling in bacterial copper amine oxidase reaction.
A key step decisively affecting the catalytic efficiency of copper amine oxidase is stereospecific abstraction of substrate alpha-proton by a conserved Asp residue. We analyzed this step by pre-steady-state kinetics using a bacterial enzyme and stereospecifically deuterium-labeled substrates, 2-phenylethylamine and tyramine. A small and temperature-dependent kinetic isotope effect (KIE) was observed with 2-phenylethylamine, whereas a large and temperature-independent KIE was observed with tyramine in the alpha-proton abstraction step, showing that this step is driven by quantum mechanical hydrogen tunneling rather than the classical transition-state mechanism. Furthermore, an Arrhenius-type preexponential factor ratio approaching a transition-state value was obtained in the reaction of a mutant enzyme lacking the critical Asp. These results provide strong evidence for enzyme-enhanced hydrogen tunneling. X-ray crystallographic structures of the reaction intermediates revealed a small difference in the binding mode of distal parts of substrates, which would modulate hydrogen tunneling proceeding through either active or passive dynamics.
Murakawa
Takeshi
T
Department of Structural Molecular Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.
Okajima
Toshihide
T
Kuroda
Shun'ichi
S
Nakamoto
Takuya
T
Taki
Masayasu
M
Yamamoto
Yukio
Y
Hayashi
Hideyuki
H
Tanizawa
Katsuyuki
K
D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry* / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D003903:Deuterium / Q000378:metabolism*; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D010084:Oxidation-Reduction; D010627:Phenethylamines / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011789:Quantum Theory; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
C086758:2,4,5-trihydroxyphenylalanine quinone; D001426:Bacterial Proteins; D010627:Phenethylamines; D004295:Dihydroxyphenylalanine; D003903:Deuterium; D006631:Amine Oxidase (Copper-Containing)