SUBSTRATE-INDUCED ACTIVATION OF DIENELACTONE HYDROLASE - AN ENZYME WITH A NATURALLY-OCCURRING CYS-HIS-ASP TRIAD

The Cys-His-Asp catalytic triad found in dienelactone hydrolase (DLH) is unusual for several reasons. It has not been observed in other hydr olytic enzymes and it is virtually inactive when it is produced by sit e-directed mutagenesis in the proteases. We propose a model to explain why this triad is catalytically active in DLH but not in the protease s. In the resting state of DLH, His2O2 forms an ion pair with Asp171 a nd Cys123 exists as a thiol. The resting state thiol does not interact with His2O2 in the active site but instead forms a hydrogen bond with Glu36 in the interior of the molecule. In the absence of substrate, G lu36 is also ion paired with Arg206. When substrate binds, Arg206 form s a second ion pair with the anionic substrate and the Arg206/Glu36 io n pair weakens. The destabilized Glu36 carboxylate shifts towards and deprotonates the Cys123 thiol, thereby activating the nucleophile. As the thiolate anion is not energetically favoured in the hydrophobic in terior of the enzyme, it swings into the active site where it can be s tabilized by the His2O2 imidazolium and the dipole of helix C. The Cys 123 thiolate which now lies adjacent to the acyl carbon of the substra te, is thus generated only in the presence of substrate. The mode of t hiolate activation reduces the susceptibility of DLH towards thiol alk ylating agents.