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 subunit. The CpcEF bilin addition activity exhibits simple Michaelis-Menten kinetics with respect to the apo-alpha(PC) and to bilin. Cpc(EF) also catalyzes the addition of phycoerythrobilin to apo-alpha(PC); phycoerythrobilin is thought to be on the biosynthetic pathway of phycocyanobilin. Cpc(EF) 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)