The catalytic function of bovine lysyl oxidase in the absence of copper.
Bovine lysyl oxidase (BLO) contains two different cofactors, copper (Kagan, H. M. (1986) in Biology of Extracellular Matrix (Mecham, R. P., ed) Vol. 1, pp. 321-398, Academic Press, Orlando, FL) and lysine tyrosyl quinone (LTQ) (Wang, S. X., Mure, M., Medzihradszky, K. F., Burlingame, A. L., Brown, D. E., Dooley, D. M., Smith, A. J., Kagan, H. M., and Klinman, J. P. (1996) Science 273, 1078-1084). By a combination of UV-visible spectroscopy, metal content analysis, and activity measurements, we find that copper-depleted BLO reacts in an irreversible manner with phenylhydrazine, an amine substrate analog, and catalyzes multiple turnovers of the substrate benzylamine. After removal of the majority of enzyme-bound copper, apoBLO exhibits a decrease in the LTQ content, as evidenced by the drop of the 510-520-nm absorbance, suggesting that the copper may play a structural role in stabilizing the LTQ. The remaining intact LTQ in the apoBLO reacted with phenylhydrazine, both in the presence and absence of the chelator, 10 mm 2,2'-dipyridyl. When benzylamine was used as the substrate, the apoBLO turned over at a rate of 50-60% of the native BLO (after correction for the residual copper and the change of LTQ content). Copper contamination from the assay buffer was ruled out by comparison of enzyme activity using different apoBLO concentrations. These studies demonstrate that the mature form of lysyl oxidase retains many of its functions in the absence of copper.
Tang
C
C
Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
Klinman
J P
JP
D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D002384:Catalysis; D002417:Cattle; D003300:Copper / Q000502:physiology*; D010659:Phenylhydrazines / Q000378:metabolism; D011487:Protein Conformation; D008249:Protein-Lysine 6-Oxidase / Q000737:chemistry / Q000378:metabolism*
D001051:Apoenzymes; D010659:Phenylhydrazines; C030299:phenylhydrazine; D003300:Copper; D008249:Protein-Lysine 6-Oxidase