title: The kinetics of ox kidney biliverdin reductase in the pre-steady state. Evidence that the dissociation of bilirubin is the rate-determining step.
abstract: When the production of bilirubin by biliverdin reductase was monitored at 460 nm by stopped-flow spectrophotometry a 'burst' was observed with a first-order rate constant at pH 8 of 20 s-1. The steady-state rate was established on completion of the 'burst'. When the reaction was monitored at 401 nm there was no observed steady-state rate, but a diminished pre-steady-state 'burst' reaction was still seen with a rate constant of 22 s-1. We argue that the rate-limiting reaction is the dissociation of bilirubin from an enzyme.NADP+.bilirubin complex. With NADPH as the cofactor the hydride-transfer step was shown to exhibit pH-dependence associated with an ionizing group with a pK of 7.2. The kinetics of NADPH binding to the enzyme at pH 7.0 were measured by monitoring the quenching of protein fluorescence on binding the coenzyme.
authors.affiliation: Department of Biochemistry, Trinity College, Dublin, Ireland.
authors.forename: E
authors.initials: E
authors.lastname: Rigney
authors.forename: T J
authors.initials: TJ
authors.lastname: Mantle
authors.forename: F M
authors.initials: FM
authors.lastname: Dickinson
mesh_terms: D000818:Animals; D001663:Bilirubin / Q000378:metabolism*; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors
chemical_list: D009249:NADP; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C019789:biliverdin reductase; D001663:Bilirubin