NATURAL MUTAGENESIS STUDY OF THE HUMAN STEROID 5-ALPHA-REDUCTASE-2 ISOZYME

The enzyme steroid 5 alpha-reductase utilizes NADPH to reduce the doub le bonds of a variety of steroid substrates with generalized 3-oxo, De lta(4,5) structures. One substrate for this membrane-bound enzyme is t estosterone, whose reduction to dihydrotestosterone is required for th e embryonic differentiation of the external male genitalia and prostat e. There are two 5 alpha-reductase isozymes, designated types 1 and 2, which have different biochemical and pharmacological properties. Inhe rited deficiencies of 5 alpha-reductase type 2 result in a form of mal e pseudohermaphroditism in which the external genitalia fail to develo p normally. Here, nine additional mutations in the 5 alpha-reductase 2 gene in subjects with 5 alpha-reductase deficiency are described. The biochemical consequences of these mutations, as well as 13 previously identified missense mutations, were characterized by recreating the m utations in an expressible cDNA and transfecting into mammalian cells. Twelve of the 22 mutations inactivated the enzyme. The remaining 10 m utations impaired enzyme function by affecting either substrate or cof actor binding. Almost all mutations decreased the half-life of the pro tein, and all but one of the impaired enzymes had an altered pH optimu m. The mutations provide insight into functional domains in the protei n as well as an unusual acidic pH optimum characteristic of the 5 alph a-reductase type 2 isozyme.