title: Catalytic activity of the D38A mutant of 3-oxo-Delta 5-steroid isomerase: recruitment of aspartate-99 as the base.
abstract: 3-oxo-Delta(5)-steroid isomerase (KSI) from Comamonas (Pseudomonas) testosteroni catalyzes the isomerization of beta,gamma-unsaturated 3-oxosteroids to their conjugated isomers through an intermediate dienolate. Residue Asp-38 (pK(a) 4.57) acts as a base to abstract a proton from C-4 of the substrate to form an intermediate dienolate, which is then reprotonated on C-6. Both Tyr-14 (pK(a) 11.6) and Asp-99 (pK(a) >/= 9.5) function 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 basic Asn (D38N) has previously been shown to result in a >10(8)-fold decrease of catalytic activity. In this work, we describe the preparation and kinetic analysis of the Ala-38 (D38A) mutant. Unexpectedly, D38A has a catalytic 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 studies 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 enzyme, which are assigned to Asp-99 and either Tyr-14 or Tyr-55. A mechanism for D38A is proposed in which Asp-99 is recruited as the catalytic base, with stabilization of the intermediate dienolate ion and the flanking transition states provided by hydrogen bonding from both Tyr-14 and Tyr-55. This mechanism is supported by the lack of detectable activity of the D38A/D99N, D38A/Y14F, and D38A/Y55F double mutants.
authors.affiliation: Laboratory for Chemical Dynamics, Department of Chemistry and Biochemistry, 1000 Hilltop Circle, University of Maryland, Baltimore County, Baltimore, Maryland 21250, USA.
authors.forename: F
authors.initials: F
authors.lastname: Hénot
authors.forename: R M
authors.initials: RM
authors.lastname: Pollack
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D020604:Comamonas testosteroni / Q000201:enzymology / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004855:Equilenin / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D013056:Spectrophotometry, Ultraviolet; D013251:Steroid Isomerases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017186:Titrimetry
chemical_list: D001224:Aspartic Acid; D013251:Steroid Isomerases; C022660:steroid delta-isomerase; D000409:Alanine; D004855:Equilenin