Abstract
The reaction and inhibition kinetics of rabbit lens aldose reductase were investigated. The kinetic reaction mechanism of the enzyme is compatible with an ordered Bi Bi reaction mechanism in which reduced nicotinamide adenine dinucleotide phosphate (NADPH) binds first to the enzyme, before glyceraldehyde, and nicotinamide adenine dinucleotide phosphate (NADP+) is released last. The Michaelis constants for NADPH and glyceraldehyde were 1.7×10-5 and 8.9×10-5M, respectively, and the dissociation constant of NADPH was 7.5×10-6M. The inhibition constant for NADP+ as a product was 1.5×10-5M. Benzyl 4-isopropyl-5-phenyl-2-oxazolecarbamate (4-isopropyl-BPOC), a potent aldose reductase inhibitor, exhibited a linear un-competitive inhibition with respect to NADPH, and a linear non-competitive inhibition with respect to glyceraldehyde. It was suggested that 4-isopropyl-BPOC binds to the enzyme-NADPH binary complex but not to the free enzyme. The inhibition constants of the inhibitor for NADPH and glyceraldehyde were 4.3×10-7 and 3.6×10-7M, respectively. The inhibitory effect of 4-isopropyl-BPOC on aldose reductase was reduced when the enzyme was treated with pyridoxal 5-phosphate. From this fact, it may be speculated that 4-isopropyl-BPOC interacts with a lysine residue in the enzyme molecule.
