SERINES AT THE ACTIVE-SITE OF 11-BETA-HYDROXYSTEROID DEHYDROGENASE TYPE-I DETERMINE THE RATE OF CATALYSIS

Short chain alcohol dehydrogenases have an invariant YXXXK motif at th e active site. Database analysis of 116 superfamily members showed tha t 92% also contain a Serine or Threonine residue at the Y+1 or Y+3 pos itions, a pattern we previously described as the ST rule. In the prese nt study we have mutated Serines in the active site, YSASK, motif of 1 1 beta-hydroxysteroid dehydrogenase type I (11 beta HSD1). These studi es were facilitated by the generation of a new specific polyclonal ant ibody (RAH113) raised against a synthetic peptide derived from the rat 11 beta-HSD1 sequence. Immunopurified RAH113 recognized a single band at 34kDa in liver homogenates. Kinetic analysis of equivalent amounts of wild type and mutant proteins showed that mutagenesis of active si te Serines resulted in modest increases of Km values for corticosteron e from 325 nM for 11 beta HSD1 to 512nM-588nM 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 c onstants with mutants displaying 10%, 1% and 1% of the wild type activ ity, respectively. When the oxidoreductase reaction was studied in who le cells mutagenesis again had a minimal effect on the Km value but dr amatically lowered first order rate constants to 34%, 5% and 6%, respe ctively, 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 subuni ts. (C) 1998 Academic Press.