Essential tyrosine residues in 3-ketosteroid-Delta(1)-dehydrogenase from Rhodococcus rhodochrous

Tetranitromethane treatment of 3-ketosteroid-Delta(1)-dehydrogenase of Rhod ococcus rhodochrous caused loss of the catalytic activity in a time- and co ncentration-dependent manner. Peptides (P-81) and (PN-83) were isolated fro m tryptic digests of the native and tetranitromethane-treated enzyme protei ns, respectively. PN-83 was the nitrated form of P-81. The amino acid seque nce was GGAPLIDYLESDDDLEFMVYPDYFGK (positions 97-124 of the dehydrogenase s equence). PN-83 showed a low yield of PTH-Tyr of position 116, i.e. less th an 5% of that of P-81, and instead a high yield of PTH-3-nitrotyrosine. Thi s indicated that tetranitromethane modifies Y-116 under the experimental co nditions used. Mutation of Y-104, Y-116, and Y-121 to smaller amino acid re sidues, Phe, Ser, or Ale, significantly changed the catalytic activity of t he dehydrogenase. All of the mutants contained FAD and exhibited the same s pectrophotometric properties as those of the wild type enzyme. The K-m, val ues for 4-androstene-3,17-dione of the Y-104, Y-116, and Y-121 mutants chan ged to large values. The most drastic change was observed for Y116A. The K- d, values for 1,4-androstadiene-3,17-dione of the Y116 mutants changed to 1 .5-2.6-fold larger values than that of the recombinant enzyme. The Y-121 mu tant enzymes exhibited catalytic activities like those of the recombinant e nzyme, but the catalytic efficiencies of Y121F and Y121A drastically decrea sed to 0.014-0.054% of that of the recombinant enzyme. The present results indicate that Y-121 plays an important role in the catalytic function, and that Y-116 and Y-104, act on binding of the substrate steroid.