Expression of human aldose and aldehyde reductases. Site-directed mutagenesis of a critical lysine 262.
Human aldose reductase (EC 1.1.1.21) and aldehyde reductase (EC 1.1.1.2) are implicated in the development of diabetic complications by a variety of mechanisms, and a number of drugs to inhibit these enzymes have been proposed for the therapy and prevention of these complications. To probe the structure and function of these two enzymes, we used site-directed mutagenesis in the cDNAs of both enzymes to replace lysine 262 with methionine. Wild-type and mutant enzymes were overexpressed in Escherichia coli and purified by anion exchange and affinity chromatography. N-terminal sequence analysis, Western blots, and kinetic studies confirmed the identity of the recombinant wild-type enzymes with the native human placental and liver enzymes. Recombinant aldose reductase (hAR) and aldehyde reductase (hGR) have apparent kinetic constants virtually identical to their respective native enzymes. The mutant aldose reductase (hARK262 greater than M) shows a 66-fold increase in Km for NADPH with respect to the wild type (1.9 +/- 0.4 microM versus 125 +/- 14 microM), whereas the Km for DL-glyceraldehyde increased 35-fold (20 +/- 2 versus 693 +/- 41 microM). The same constants for the mutant aldehyde reductase (hGRK262 greater than M) increased 97- and 86-fold, respectively (from 2.0 +/- 0.4 to 194 +/- 16 microM and from 1.6 +/- 0.4 to 137 +/- 3 mM). These results indicate that lysine 262 in aldose reductase and aldehyde reductase is crucial to their catalytic activity by affecting co-factor binding.
Bohren
K M
KM
Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030.
Page
J L
JL
Shankar
R
R
Henry
S P
SP
Gabbay
K H
KH
D000449:Aldehyde Reductase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008094:Lithium / Q000494:pharmacology; D018020:Lithium Compounds; D008239:Lysine; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013431:Sulfates / Q000494:pharmacology
D018020:Lithium Compounds; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013431:Sulfates; C054097:lithium sulfate; D008094:Lithium; D000449:Aldehyde Reductase; D008239:Lysine