An examination of the copper requirement of phenylalanine hydroxylase from Chromobacterium violaceum.
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 microM, 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Carr
R T
RT
Department of Chemistry, Pennsylvania State University, University Park 16802.
Benkovic
S J
SJ
D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D002614:Chelating Agents / Q000494:pharmacology; D002861:Chromobacterium / Q000201:enzymology*; D003300:Copper / Q000737:chemistry; D008667:Metalloproteins / Q000737:chemistry; D010651:Phenylalanine Hydroxylase / Q000737:chemistry*; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D013057:Spectrum Analysis; D014364:Tryptophan / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
D001426:Bacterial Proteins; D002614:Chelating Agents; D008667:Metalloproteins; D011994:Recombinant Proteins; D003300:Copper; D014364:Tryptophan; D010651:Phenylalanine Hydroxylase; D015032:Zinc