Characterization of the reduction of 3-acetylpyridine adenine dinucleotide phosphate by benzyl alcohol catalyzed by aldose reductase.
Aldose reductase from rat lens catalyzes reduction of the 3-acetylpyridine analog of NADP+ by benzyl alcohol; the fluorescence of the reduced pyridine nucleotide produced in this reaction forms the basis of a new, more sensitive assay for this enzyme. The pH dependence and kcat/Km ratio of this enzymatic reaction are very similar with NADP+ or the analog; however, at pH 7.5, the reaction with the analog has a sevenfold greater kcat. In an NADPH-supported reduction reaction, aldose reductase exhibited similar activity toward benzaldehyde and glyceraldehyde, but benzyl alcohol was a much better substrate than physiological polyols in the analog-dependent oxidation reaction: relative kcat/Km ratios were 740 for benzyl alcohol, 2.2 for xylitol, and 1.0 for glycerol. By this assay, a new, high-affinity aldose reductase inhibitor AL-1567 showed noncompetitive inhibition with the pyridine nucleotide analog, with Ki = 2.05 X 10(-6) M, and competitive inhibition with benzyl alcohol, with Ki = 5.7 X 10(-9) M, indicating that the inhibitor binds the free enzyme with extremely high affinity. Compared to spectrophotometric assays of aldose reductase activity, the fluorometric assay extends the lower limit of detectability of enzyme activity by a factor of 100, and allows the determination of Ki values for potent inhibitors of the enzyme with greater accuracy.
Griffin
B W
BW
McNatt
L G
LG
D000438:Alcohols / Q000378:metabolism; D000449:Aldehyde Reductase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism; D000818:Animals; D019905:Benzyl Alcohol; D001592:Benzyl Alcohols / Q000378:metabolism*; D001593:Benzyl Compounds / Q000378:metabolism*; D002384:Catalysis; D005449:Fluorenes / Q000494:pharmacology; D006827:Hydantoins / Q000494:pharmacology; D007700:Kinetics; D007908:Lens, Crystalline / Q000201:enzymology; D009249:NADP / Q000031:analogs & derivatives* / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013401:Sugar Alcohol Dehydrogenases / Q000378:metabolism*
D000438:Alcohols; D000447:Aldehydes; D001592:Benzyl Alcohols; D001593:Benzyl Compounds; D005449:Fluorenes; D006827:Hydantoins; C048483:3-acetylpyridine-adenine dinucleotide phosphate; C045887:alconil; D009249:NADP; D013401:Sugar Alcohol Dehydrogenases; D000449:Aldehyde Reductase; D019905:Benzyl Alcohol