Characterization of phycoviolobilin phycoerythrocyaninâα84âcysteinâlyaseâ(isomerizing) from Mastigocladus laminosus
2002
Zhao, KaiâHong | Wu, Dong | Wang, Lu | Zhou, Ming | Storf, Max | Bubenzer, Claudia | Strohmann, Brigitte | Scheer, Hugo
Cofactor requirements and enzyme kinetics have been studied of the novel, dualâaction enzyme, the isomerizing phycoviolobilin phycoerythrocyaninâα84âcysteinâlyase(PVBâPECâlyase) from Mastigocladus laminosus, which catalyses both the covalent attachment of phycocyanobilin to PecA, the apoâαâsubunit of phycoerythrocyanin, and its isomerization to phycoviolobilin. Thiols and the divalent metals, Mg2+ or Mn2+, were required, and the reaction was aided by the detergent, Triton Xâ100. Phosphate buffer inhibits precipitation of the proteins present in the reconstitution mixture, but at the same time binds the required metal. Kinetic constants were obtained for both substrates, the chromophore (Kmâ=â12–16âµm, depending on [PecA], kcatâ≈â1.2â×â10−4·s−1) and the apoprotein (Kmâ=â2.4âµm at 14âµm PCB, kcatâ=â0.8â×â10−4·s−1). The kinetic analysis indicated that the reconstitution reaction proceeds by a sequential mechanism. By a combination of untagged and Hisâtagged subunits, evidence was obtained for a complex formation between PecE and PecF (subunits of PVBâPECâlyase), and by experiments with single subunits for the prevalent function of PecE in binding and PecF in isomerizing the chromophore.
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