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Biliverdin-IXareductase fromSynechocystisPCC6803 (sBVR-A) is a stable dimer and this behaviour is observed under a range of conditions. This is in contrast to all other forms of BVR-A, which have been reported to behave as monomers, and places sBVR-A in the dihydrodiol dehydrogenase⁄N-ter-minally truncated glucose–fructose oxidoreductase structural family of dimers. | The effect of pH on the initial rate kinetics of the dimeric biliverdin-IXa reductase from the cyanobacterium Synechocystis PCC6803 Jerrard M. Hayes and Timothy J. Mantle Schoolof Biochemistry and Immunology Trinity College Dublin Ireland Keywords biliverdin reductase compulsory ordered mechanism dimer pH Synechocystis Correspondence J. M. Hayes Schoolof Biochemistry and Immunology Trinity College Dublin 2 Ireland Fax 353 677 2400 Tel 353 895 1612 E-mail jehayes@tcd.ie Received 23 April2009 revised 9 June 2009 accepted 11 June 2009 doi 10.1111 j.1742-4658.2009.07149.x Biliverdin-IXa reductase from Synechocystis PCC6803 sBVR-A is a stable dimer and this behaviour is observed under a range of conditions. This is in contrast to all other forms of BVR-A which have been reported to behave as monomers and places sBVR-A in the dihydrodiol dehydrogenase N-ter-minally truncated glucose-fructose oxidoreductase structural family of dimers. The cyanobacterial enzyme obeys an ordered steady-state kinetic mechanism at pH 5 with NADPH being the first to bind and NADP the last to dissociate. An analysis of the effect of pH on kcat with NADPH as cofactor reveals a pK of 5.4 that must be protonated for effective catalysis. Analysis of the effect of pH on kcat KmNADPH identifies pK values of 5.1 and 6.1 in the free enzyme. Similar pK values are identified for biliverdin binding to the enzyme-NADPH complex. The lower pK values in the free enzyme pK 5.1 and enzyme-NADPH complex pK 4.9 are not evident when NADH is the cofactor suggesting that this ionizable group may interact with the 2 -phosphate of NADPH. His84 is implicated as a crucial residue for sBVR-A activity because the H84A mutant has less than 1 of the activity of the wild-type and exhibits small but significant changes in the protein CD spectrum. Binding of biliverdin to sBVR-A is conveniently monitored by following the induced CD spectrum for biliverdin. Binding of biliverdin to wild-type sBVR-A induces a P-type spectrum. .
