Rates of oxygen and hydrogen exchange as indicators of TPQ cofactor orientation in amine oxidases.
This study presents the first detailed examination by resonance Raman (RR) spectroscopy of the rates of solvent exchange for the C5 and C3 positions of the TPQ cofactor in several wild-type copper-containing amine oxidases and mutants of the amine oxidase from Hansenula polymorpha (HPAO). On the basis of crystal structure analysis and differing rates of C5 [double bond] O and C3 [bond] H exchange within the enzyme systems, but equally rapid rates of C5 [double bond] O and C3 [bond] H exchange in a TPQ model compound, it is proposed that these data can be used to determine the TPQ cofactor orientation within the active site of the resting enzyme. A rapid rate of C5 [double bond] O exchange (t(1/2) < 30 min) and a slow (t(1/2) = 6 h) to nonexistent rate of C3 [bond] H exchange was observed for wild-type HPAO, the amine oxidase from Arthrobacter globiformis, pea seedling amine oxidase at pH 7.1, and the E406Q mutant of HPAO. This pattern is ascribed to a productive TPQ orientation, with the C5 [double bond] O near the substrate-binding site and the C3 [bond] H near the Cu. In contrast, a slow rate of C5 [double bond] O exchange (t(1/2) = 1.6-3.3 h) coupled with a fast rate of C3 [bond] H exchange (t(1/2) < 30 min) was observed for the D319E and D319N catalytic base mutants of HPAO and for PSAO at pH 4.6 (t(1/2) = 4.5 h for C5 [double bond] O exchange). This pattern identifies a flipped orientation, involving 180 degrees rotation about the C alpha-C beta bond, which locates the C3 [bond] H near the substrate-binding site and the C5 double bond] O near the Cu. Finally, fast rates of both C5 [double bond] O and C3 [bond] H exchange (t(1/2) < 30 min) were observed for the amine oxidase from Escherichia coli and the N404A mutant of HPAO, suggesting a mobile cofactor, with multiple TPQ orientations between productive and flipped. These results demonstrate that opposing sides of the TPQ ring possess different degrees of solvent accessibility and that the rates of C5 [double bond] O and C3 [bond] H exchange can be used to predict the TPQ cofactor orientation in the resting forms of these enzymes.
Green
Edward L
EL
Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering, Oregon Health and Science University, Beaverton, Oregon 97006-8921, USA.
Nakamura
Nobuhumi
N
Dooley
David M
DM
Klinman
Judith P
JP
Sanders-Loehr
Joann
J
D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D009154:Mutation; D010100:Oxygen / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology; D011485:Protein Binding; D013059:Spectrum Analysis, Raman
C086758:2,4,5-trihydroxyphenylalanine quinone; D004295:Dihydroxyphenylalanine; D006859:Hydrogen; D006631:Amine Oxidase (Copper-Containing); D010100:Oxygen