Inhibition of aldose reductase by (2,6-dimethylphenylsulphonyl)nitromethane: possible implications for the nature of an inhibitor binding site and a cause of biphasic kinetics.
Aldose reductase (aldehyde reductase 2, ALR2) is often isolated as a mixture of two forms which are sensitive (ALR2S), or insensitive (ALR2I), to inhibitors. We show that ICI 215918 ((2-6-dimethylphenylsulphonyl)-nitromethane) follows either noncompetitive, or uncompetitive kinetics with respect to aldehyde for ALR2S, or the closely related enzyme, aldehyde reductase (aldehyde reductase 1, ALR1). Similar behaviour is exhibited by two other structural types of aldose reductase inhibitor (ARI), spirohydantoins and acetic acids, when either aldehyde, or NADPH is varied. For ALR2S, we have demonstrated kinetic competition between a sulphonylnitromethane, an acetic acid and a spirohydantoin. Thus, different ARIs probably have overlapping binding sites. Published studies imply that ALR2 follows an ordered mechanism where coenzyme binds first and induces a reversible conformation change (E.NADPH-->E*.NADPH). Reduction of aldehyde appears rate-limited by the step E*.NADP+-->E.NADP+. Spontaneous activation converts ALR2S into ALR2I and increases kcat. This must be associated with acceleration of the rate-determining step. We now propose the following hypothesis to explain characteristics of ARIs. (1) Inhibitors preferentially bind to the E* conformation. (2) The ARI binding site contains residues in common with that for aldehyde substrates. When aldehyde is varied, uncompetitive inhibition arises from association at the site for alcohol product in the E*.NADP+ complex which has little affinity for the substrate. Any competitive inhibition arises from use of the aldehyde site in the E*.NADPH complex. (3) Acceleration of the E*.NADP+-->E.NADP+ step upon activation of ALR2 reduces steady state levels of E* and so decreases sensitivity to ARIs.
ICI Pharmaceuticals, Macclesfield, Cheshire, U.K.
W H
WH
Ward
P N
PN
Cook
D J
DJ
Mirrlees
D R
DR
Brittain
J
J
Preston
F
F
Carey
D P
DP
Tuffin
R
R
Howe
D000085:Acetates / Q000494:pharmacology; D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D006801:Humans; D006827:Hydantoins / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008956:Models, Chemical; D009595:Nitroparaffins / Q000494:pharmacology*; D013450:Sulfones / Q000494:pharmacology*
D000085:Acetates; D006827:Hydantoins; D009595:Nitroparaffins; D013450:Sulfones; C071862:(2,6-dimethylphenylsulfonyl)nitromethane; D000449:Aldehyde Reductase