tailieunhanh - Báo cáo khoa học: Coenzyme A affects firefly luciferase luminescence because it acts as a substrate and not as an allosteric effector

The effect of CoA on the characteristic light decay of the firefly luciferase catalysed bioluminescence reaction was studied. At least part of the light decay is due to the luciferase catalysed formation of dehydroluciferyl-adenylate (L-AMP), a by-product that results from oxidation of luciferyl-adenylate (LH2-AMP), and is a powerful inhibitor of the bioluminescence reaction (IC50¼6nm). | iFEBS Journal Coenzyme A affects firefly luciferase luminescence because it acts as a substrate and not as an allosteric effector Hugo Fraga1 2 Diogo Fernandes1 2 Rui Fontes2 and Joaquim C. G. Esteves da Silva1 1 Departmento de Quimica Faculdade de Ciencias da Universidade do Porto Portugal 2 Servico de Bioquimica U38-FCT Faculdade de Medicina da Universidade do Porto Portugal Keywords Coenzyme A dehydroluciferyl-adenylate dehydroluciferyl-coenzyme A dephosphocoenzyme A firefly luciferase Correspondence J. C. G. Esteves da Silva Departmento de Quimica Faculdade de Ciencias da Universidade do Porto R. Campo Alegre 687 4169-007 Porto Portugal Fax 351 226082959 Tel 351 226082869 E-mail jcsilva@ Received 24 May 2005 revised 28 June 2005 accepted 18 July 2005 doi The effect of CoA on the characteristic light decay of the firefly luciferase catalysed bioluminescence reaction was studied. At least part of the light decay is due to the luciferase catalysed formation of dehydroluciferyl-adenylate L-AMP a by-product that results from oxidation of luciferyl-adenylate LH2-AMP and is a powerful inhibitor of the bioluminescence reaction IC50 6 nM . We have shown that the CoA induced stabilization of light emission does not result from an allosteric effect but is due to the thiolytic reaction between CoA and L-AMP which gives rise to dehydro-luciferyl-CoA L-CoA a much less powerful inhibitor IC50 5 pM . Moreover the Vmax for L-CoA formation was determined as 160 min-1 which is one order of magnitude higher than the Vmax of the bioluminescence reaction. Results obtained with CoA analogues also support the thiolytic reaction mechanism CoA analogues without the thiol group dethio-CoA and acetyl-CoA do not react with L-AMP and do not antagonize its inhibitor effect CoA and dephospho-CoA have free thiol groups both react with L-AMP and both antagonize its effect. In the case of dephospho-CoA it was shown that it reacts with L-AMP forming .

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