The effect of pH on the initial rate kinetics of the dimeric biliverdin-IXalpha reductase from the cyanobacterium Synechocystis PCC6803.
Biliverdin-IXalpha 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-terminally 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 k(cat) with NADPH as cofactor reveals a pK of 5.4 that must be protonated for effective catalysis. Analysis of the effect of pH on k(cat)/K(m)(NADPH) 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. The H84A mutant shows evidence for weak binding of biliverdin and appears to bind a variant of the P-configuration. Intriguingly, the Y102A mutant, which is catalytically active, binds biliverdin in the M-configuration.
Hayes
Jerrard M
JM
School of Biochemistry and Immunology, Trinity College, Dublin, Ireland. jehayes@tcd.ie
Mantle
Timothy J
TJ
D001664:Biliverdine / Q000378:metabolism; D000458:Cyanobacteria / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D050505:Mutant Proteins / Q000378:metabolism; D020125:Mutation, Missense; D009249:NADP; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D046939:Synechocystis / Q000201:enzymology*
D050505:Mutant Proteins; D009249:NADP; D044925:Oxidoreductases Acting on CH-CH Group Donors; C019789:biliverdin reductase; D001664:Biliverdine