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Published November 14, 1997 | Published
Journal Article Open

Characterization of the Native Lysine Tyrosylquinone Cofactor in Lysyl Oxidase by Raman Spectroscopy

Abstract

Lysine tyrosylquinone (LTQ) recently has been identified as the active site cofactor in lysyl oxidase by isolation and characterization of a derivatized active site peptide. Reported in this study is the first characterization of the underivatized cofactor in native lysyl oxidase by resonance Raman (RR) spectrometry. The spectrum is characterized by a unique set of vibrational modes in the 1200 to 1700 cm^(−1) region. We show that the RR spectrum of lysyl oxidase closely matches that of a synthetic LTQ model compound, 4-n-butylamino-5-ethyl-1,2-benzoquinone, in aqueous solutions but differs significantly from those of other topa quinone-containing amine oxidases under similar conditions. Furthermore, we have observed the same ^(18)O shift of the C=O stretch in both the lysyl oxidase enzyme and the LTQ cofactor model compound. The RR spectra of different model compounds and their D shifts give additional evidence for the protonation state of LTQ cofactor in the enzyme. The overall similarity of these spectra and their shifts shows that the lysyl oxidase cofactor and the model LTQ compound have the same structure and properties. These data provide strong and independent support for the new cofactor structure, unambiguously ruling out the possibility that the structure originally reported had been derived from a spurious side reaction during the derivatization of the protein and isolation of the active site peptide.

Additional Information

© 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Received for publication, September 3, 1997, and in revised form, September 23, 1997. This work was supported by National Institutes of Health Grants GM-39296 (to J. P. K.) and GM-34468 (to J. S.-L.), as well as support from the W. M. Keck Foundation (to J. P. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. We are grateful to Dr. David M. Dooley and Doreen E. Brown for the sample of pea seedling amine oxidase. We also want to thank Hitoshi Yamada at ISIR, Osaka University for the assistance in mass spectrometric analysis of the model compounds.

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