Abstract
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.
Highlights
Protein-lysine 6-oxidase1 is an extracellular matrix protein
By a combination of UV-visible spectroscopy, metal content analysis, and activity measurements, we find that copper-depleted bovine lysyl oxidase (BLO) reacts in an irreversible manner with phenylhydrazine, an amine substrate analog, and catalyzes multiple turnovers of the substrate benzylamine
It has been assigned to the copper amine oxidase family based on the presence of copper and its catalysis of the oxidative deamination of amine to aldehyde [1, 2]
Summary
Bovine lysyl oxidase (BLO) contains two different cofactors, copper 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. The lysine tyrosyl quinone (LTQ) has been identified as the active site carbonyl cofactor in bovine lysyl oxidase [7]. It is observed that BLO retains its ability to interact with amines in the absence of copper These results are related to the studied role for copper in the analogous 2,4,5-trihydroxyphenylalanine-containing copper amine oxidases [8, 9]. We propose that the established physiological requirement for copper in LO [3] is a consequence of an essential role for copper in LTQ biogenesis, together with a role for copper in the maintenance of cofactor and/or protein structural integrity
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