Catalytic activity of the D38A mutant of 3-oxo-Delta(5)-steroid isomerase:Recruitment of aspartate-99 as the base

3-Oxo-Delta(5)-steroid isomerase (KSI) from Comamonas (Pseudomonas) testost eroni catalyzes the isomerization of beta,gamma-unsaturated 3-oxosteroids t o their conjugated isomers through an intermediate dienolate. Residue Asp-3 8 (pK(a) 4.57) acts as a base to abstract a proton from C-4 of the substrat e to form an intermediate dienolate, which is then reprotonated on C-6. Bot h Tyr-14 (pK(a) 11.6) and Asp-99 (pK(a) greater than or equal to 9.5) funct ion as hydrogen-bond donors to O-3 of the steroid, helping to stabilize the transition states. Mutation of the active-site base Asp-38 to the weakly b asic Asn (D38N) has previously been shown to result in a > 10(8)-fold decre ase of catalytic activity. In this work, we describe the preparation and ki netic analysis of the Ala-38 (D38A) mutant. Unexpectedly, D38A has a cataly tic turnover number (k(cat)) that is ca. 10(6)-fold greater than the value for D38N and only about 140-fold less than that for wild type. Kinetic stud ies as a function of pH show that D38A-catalyzed isomerization involves two groups, with pK(a) values of 4.2 and 10.4, respectively, in the free enzym e, which are assigned to Asp-99 and either Tyr-14 or Tyr-55. A mechanism fo r D38A is proposed in which Asp-99 is recruited as the catalytic base, with stabilization of the intermediate dienolate ion and the flanking transitio n states provided by hydrogen bonding from both Tyr-14 and Tyr-55. This mec hanism is supported by the lack of detectable activity of the D38A/D99N, D3 8A/Y14F, and D38A/Y55F double mutants.