An Examination of the Copper Requirement of Phenylalanine Hydroxylase from Chromobacterium violaceum

Robert T. Carr, Stephen Benkovic

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Phenylalanine hydroxylase from Chromobacterium violaceum (CVPAH) was classified as a copper metalloenzyme by virtue of a 1/1 Cu/enzyme stoichiometry and its inhibition with various chelators [Pember, S. O., Villafranca, J. J., & Benkovic, S. J. (1986) Biochemistry 25, 6611]. We have prepared “copper-free” CVPAH by extraction with DTT. These preparations retained full activity though the Cu/enzyme ratio averaged 0.015. Reconstitution by extraneous copper was disproved by measuring a Cu/enzyme ratio of 0.09 in assay mixtures after the specific activity was determined to be within 85% of a fully copper-complexed control. Several copper chelators were examined and were not inhibitory. The “copper-free” enzyme had significant activity without a thiol or other reducing agent capable of reducing the copper center whereas copper-complexed CVPAH had minimal activity under these conditions. Copper-complexed CVPAH can be activated, however, by nonreducing copper ligands such as imidazole. From these results, we conclude that copper is not a requirement of activity. Iron, cobalt, nickel, manganese, molybdenum, and chromium were not found in the “copper-free” preparation, indicating that the active hydroxylating species may not require a redox-active metal. The Kds for binding Cu2+ and Zn2+ were measured to be 0.48 and 0.85 µM, respectively. Both copper and zinc were found to be potent inhibitors of “copper-free” CVPAH in the absence of thiols. DTT reverses inhibition due to Cu2+ but not inhibition caused by Zn2+. The product stoichiometry indicates the same tightly coupled turnover found with all other pterin-dependent hydroxylases when using natural substrates. The relevance of these results to mammalian pterin-requiring hydroxylases is discussed in terms of the metal requirement for the formation of active oxygen intermediates.

Original languageEnglish (US)
Pages (from-to)14132-14138
Number of pages7
Issue number51
StatePublished - 1993

All Science Journal Classification (ASJC) codes

  • Biochemistry


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