Serines at the active site of 11 beta-hydroxysteroid dehydrogenase type I determine the rate of catalysis
| Autor: | Zygmunt S. Krozowski, Kevin X. Z. Li, Wieslaw H. Trzeciak, Varuni R. Obeyesekere |
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| Rok vydání: | 1998 |
| Předmět: |
Mutant
Biophysics Dehydrogenase Transfection Biochemistry Serine Oxidoreductase Animals Threonine Molecular Biology chemistry.chemical_classification Short-chain dehydrogenase Binding Sites biology Chemistry Wild type Hydroxysteroid Dehydrogenases Active site Cell Biology Molecular biology Rats Enzyme Activation Mutation biology.protein Mutagenesis Site-Directed 11-beta-Hydroxysteroid Dehydrogenases Peptides |
| Zdroj: | Biochemical and biophysical research communications. 250(2) |
| ISSN: | 0006-291X |
| Popis: | Short chain alcohol dehydrogenases have an invariant YXXXK motif at the active site. Database analysis of 116 superfamily members showed that 92% also contain a Serine or Threonine residue at the Y + 1 or Y + 3 positions, a pattern we previously described as the ST rule. In the present study we have mutated Serines in the active site, YSASK, motif of 11 beta-hydroxysteroid dehydrogenase type I (11 beta HSD1). These studies were facilitated by the generation of a new specific polyclonal antibody (RAH113) raised against a synthetic peptide derived from the rat 11 beta-HSD1 sequence. Immunopurified RAH113 recognized a single band at 34 kDa in liver homogenates. Kinetic analysis of equivalent amounts of wild type and mutant proteins showed that mutagenesis of active site Serines resulted in modest increases of Km values for corticosterone from 325 nM for 11 beta HSD1 to 512 nM-588 nM for the S1 (YAASK), S2 (YSAGK) and S3 (YAAGK) mutants in homogenates of transfected CHOP cells. However, far greater effects were observed on the first order rate constants with mutants displaying 10%, 1% and 1% of the wild type activity, respectively. When the oxidoreductase reaction was studied in whole cells mutagenesis again had a minimal effect on the Km value but dramatically lowered first order rate constants to 34%, 5% and 6%, respectively, of the wild type. These data show that Serines at the active site of 11 beta HSD1 play an important role in determining the rate of catalysis. Coexpression of wild type and mutant enzymes did not lower wild type activity suggesting that the active site of the multimeric enzyme is not a composite of active site Serines from different subunits. |
| Databáze: | OpenAIRE |
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