The enzyme 3-oxo-Delta5-steroid isomerase is a key enzyme in the biosynthesis of steroids. We wish to utilize the techniques of protein engineering to probe three aspects of the enzyme 3-oxo-Delta5-steroid isomerase from Pseudomonas testorsteroni: (1) the identity of the residues involved in catalysis, (2) the nature of the interaction of the active-site with steroid substrates and inhibitors, and (3) the possible catalytic contribution of hydrophobic interactions by selective binding of the transition state over the ground state. In addition we will confirm the identity of the amino acid sequence of the enzyme by direct sequencing of the gene. Potential residues to be modified are Asp-38, His-100, Asn-57, Tyr-88, Tyr-14, Phe-101, and Phe-103. The first five of these residues have been implicated in the catalytic mechanism, whereas the last two are thought to be involved in the interaction between the steroid substrate and the active site of the isomerase. Should any of the modified enzymes be inactive, binding studies will be performed to determine if the inactivation is due to inability of the mutant enzyme to bind the substrate or to impairment of the catalytic machinery. For those mutant enzymes which retain activity, kinetic studies will be performed to determine whether there is any effect of the mutation on the kinetic parameters k-cat and Km. pH-rate profiles will also be examined to determine if there is any change due to the mutation. Mutant enzymes which appear to be of particular interest will be submitted to the Argonne National Laboratory for determination of their structures by x-ray crystallography.
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