A THEORETICAL-STUDY OF SUBSTRATE-INDUCED ACTIVATION OF DIENELACTONE HYDROLASE

Dienelactone hydrolase (DLH), an enzyme from the beta-ketoadipate path way, catalyses the hydrolysis of dienelactone to maleylacetate. DLH is unusual because it is the only known naturally occurring enzyme which contains the catalytic triad Cys...His...Asp. This triad has previous ly been created artificially in the mutant serine proteases, thiol sub tilisin and thiol trypsin. In both cases the mutant enzymes exhibited activities several orders of magnitude lower than the wild type enzyme s; the low reactivity has generally been attributed to the inability o f these enzymes to form a catalytically active thiolate anion (Cys ... His(+)...Asp(-)). The crystal structure of DLH suggests that the nativ e enzyme exists predominantly in a catalytically inert configuration; the triad cysteine is neutral and points away from the active site bin ding cleft. However, a crystallographic analysis of C123S DLH complexe d with an isostructural inhibitor (dienelactam) indicates that substra te binding induces a prototropic rearrangement of the active site prio r to catalysis which results in the formation of a highly nucleophilic thiolate anion. We have performed ab initio SCF/MP2 calculations on a relatively small portion of the active site of DLH to examine the det ails of this activation process. Our calculations provide supporting e vidence that the conformational changes observed in the crystal struct ure due to inhibitor (or substrate) binding facilitate the formation o f a reactive thiolate anion. In particular, substrate binding alters t he position of Glu36; the carboxylate side chain of Glu36 is pushed to wards C123 enabling it to abstract the thiol proton thus creating a ca talytically active thiolate anion. The calculations also provide a pos sible explanation for the low reactivities observed in the mutant seri ne proteases.