Role of the tertiary structure in the diphenol oxidase activity of Octopus vulgaris hemocyanin

S. Campello, M. Beltramini, G. Giordano, P. Di Muro, S. M. Marino, L. Bubacco

Research output: Contribution to journalArticle

Abstract

The functional differences between the oxygen transport protein Hemocyanin and the enzymes Tyrosinase and Catechol oxidase are believed to be governed, at least in part, by the tertiary structure, which differs in these molecules and controls the accessibility of their copper containing active site for substrate(s). Accordingly, Octopus vulgaris Hemocyanin catalyses the o-diphenol oxidation to o-quinone at a very low rate. The crystallographic structure of one of the functional units (called Odg) of O. dofleini Hemocyanin shows two domains, a mainly α-helical domain that directly binds the copper ions of the reaction center and a β-strand domain that precludes access to the active site to ligands bigger than molecular oxygen. In this work, we have first cleaved the whole protein and then purified different oxygen binding functional units from O. vulgaris Hemocyanin. These functional units were used in activity assays with l-DOPA, the paradigmatic substrate for Catechol oxidase. All functional units show a negligible enzymatic activity. The procedure to generate the functional units induces in only one of them a proteolytic cleavage. Amino terminal sequencing and mass spectroscopy of the fragments allow to place the cleavage site between the alpha and beta domains of the functional unit homologous to Odd, in the O. dofleini sequence. An increase, up to three orders of magnitude, of Tyrosinase-like activity was observed when the cleaved Odd-like was incubated with the substrate in the presence of trifluoroethanol or hexafluoroisopropanol.

Original languageEnglish
Pages (from-to)159-167
Number of pages9
JournalArchives of Biochemistry and Biophysics
Volume471
Issue number2
DOIs
Publication statusPublished - Mar 15 2008

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Keywords

  • Catalytic activity
  • Copper
  • Hemocyanin
  • Octopus vulgaris
  • Oxygen

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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