Oligomeric structure, enzyme kinetics, and substrate specificity of the phycocyanin alpha subunit phycocyanobilin lyase.
Phycobiliproteins carry linear tetrapyrrole chromophores (bilins) thioether-linked to specific cysteine residues. The process of bilin attachment to apoprotein in vivo has been characterized for only one bilin attachment site on one phycobiliprotein, that on the alpha subunit of phycocyanin (alpha PC). In the cyanobacterium Synechococcus sp. PCC 7002, the addition of phycocyanobilin to apo-alpha PC is catalyzed by the protein products of the cpcE and cpcF genes. We have purified and further characterized the recombinant CpcE and CpcF proteins. CpcE and CpcF form an enzymatically active 1:1 complex (CpcEF), stable to size exclusion chromatography. CpcEF causes a reduction in alpha PC fluorescence and strongly affects its absorption spectrum but has no effect on the beta subunit. The CpcEF bilin addition activity exhibits simple Michaelis-Menten kinetics with respect to the apo-alpha PC and to bilin. CpcEF also catalyzes the addition of phycoerythrobilin to apo-alpha PC; phycoerythrobilin is thought to be on the biosynthetic pathway of phycocyanobilin. CpcEF shows a preference for phycocyanobilin relative to phycoerythrobilin, both in binding affinity and in the rate of catalysis, sufficient to account for selective attachment of phycocyanobilin to apo-alpha PC.
Fairchild
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Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Glazer
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D001654:Bile Pigments / Q000378:metabolism; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000201:enzymology*; D005796:Genes; D007700:Kinetics; D008190:Lyases / Q000737:chemistry*; D008970:Molecular Weight; D010798:Phycocyanin / Q000737:chemistry*; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D001654:Bile Pigments; D011994:Recombinant Proteins; D010798:Phycocyanin; D008190:Lyases; C075919:phycocyanin alpha-subunit phycocyanobilin lyase