Activation of human biliverdin-IXα reductase by urea: generation of kinetically distinct forms during the unfolding pathway.
Activation of enzymes by low concentrations of denaturants has been reported for a limited number of enzymes including lipocalin-type prostaglandin D synthase (L-PGDS) and adenylate kinase. During unfolding studies on human biliverdin-IXα reductase it was discovered that the enzyme is activated at low concentrations of urea. Under standard assay conditions the native enzyme displays pronounced substrate inhibition with biliverdin as variable substrate; however in the presence of 3M urea, the substrate inhibition is abolished and the enzyme exhibits Michaelian kinetics. When the initial rate kinetics with NADPH as variable substrate are conducted in 3M urea, the Vmax is increased 11-fold to 1.8μmol/min/mg and the apparent Km for biliverdin increases from 1 to 3μM. We report the existence of two kinetically distinct folded intermediates between the native and unfolded forms. When the period of incubation with urea was varied prior to measuring enzyme activity, the apparent Vmax was shown to decay to half that seen at zero time with a half life of 5.8minutes, while the apparent Km for NADPH remains constant at approximately 5μM. With NADH as cofactor the half life of the activated (A) form was 2.9minutes, and this form decays in 3M urea to a less active (LA) form. The apparent Km for NADH increases from 0.33mM to 2mM for the A and LA forms. These kinetically distinct species are reminiscent of the activity-enhanced and inactive forms of L-PGDS observed in the presence of urea and guanidine hydrochloride.
School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, 152-160 Pearse Street, Dublin 2, Ireland. Electronic address: efrankli@tcd.ie.
Edward
E
Franklin
Timothy
T
Mantle
Aisling
A
Dunne
D001664:Biliverdine; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D058767:Protein Unfolding; D014508:Urea / Q000737:chemistry*
D014508:Urea; D010088:Oxidoreductases; D001664:Biliverdine
Activation; Biliverdin reductase; HO-1; Mrp2; PQQ; Pyridine nucleotides; QM/MM; ROS; Substrate inhibition; UGT1A1; Unfolding; bilirubin UDP-glucuronosyltransferase 1; hBVR-A; haem oxygenase 1; human biliverdin-IXα reductase; multidrug resistance protein 2; pyrroloquinoline quinone; quantum mechanics/molecular-mechanics; reactive oxygen species; the free energy of unfolding; ΔG(u)