Mutation of threonine-241 to proline eliminates autocatalytic modification of human carbonyl reductase.
Carbonyl reductase catalyses the reduction of steroids, prostaglandins and a variety of xenobiotics. An unusual property of human and rat carbonyl reductases is that they undergo modification at lysine-239 by an autocatalytic process involving 2-oxocarboxylic acids, such as pyruvate and 2-oxoglutarate. Comparison of human carbonyl reductase with the pig enzyme, which does not undergo autocatalytic modification, identified three sites, alanine-236, threonine-241 and glutamic acid-246, on human carbonyl reductase that could be important in the reaction of lysine-239 with 2-oxocarboxylic acids. Mutagenesis experiments show that replacement of threonine-241 with proline (T241P) in human carbonyl reductase eliminates the formation of carboxyethyl-lysine-239. In contrast, the T241A mutant has autocatalytic activity similar to wild-type carbonyl reductase. The T241P mutant retains catalytic activity towards menadione, although with one-fifth the catalytic efficiency of wild-type carbonyl reductase. Replacement of threonine-241 with proline is likely to disrupt the local structure near lysine-239. We propose that integrity of this local environment is essential for chemical modification of lysine-239, but not absolutely required for carbonyl reductase activity.
Chemisches Zentrallabor, Inselspital, 3010 Berne, Switzerland.
M A
MA
Sciotti
S
S
Nakajin
B
B
Wermuth
M E
ME
Baker
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002384:Catalysis; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013552:Swine; D013737:Testis / Q000201:enzymology; D013912:Threonine / Q000235:genetics*
D013912:Threonine; D011392:Proline; D000429:Alcohol Oxidoreductases; C019642:alcohol dehydrogenase (NADP+)