title: Evaluation of the Catalytic Contribution from a Positioned General Base in Ketosteroid Isomerase.
abstract: Proton transfer reactions are ubiquitous in enzymes and utilize active site residues as general acids and bases. Crystal structures and site-directed mutagenesis are routinely used to identify these residues, but assessment of their catalytic contribution remains a major challenge. In principle, effective molarity measurements, in which exogenous acids/bases rescue the reaction in mutants lacking these residues, can estimate these catalytic contributions. However, these exogenous moieties can be restricted in reactivity by steric hindrance or enhanced by binding interactions with nearby residues, thereby resulting in over- or underestimation of the catalytic contribution, respectively. With these challenges in mind, we investigated the catalytic contribution of an aspartate general base in ketosteroid isomerase (KSI) by exogenous rescue. In addition to removing the general base, we systematically mutated nearby residues and probed each mutant with a series of carboxylate bases of similar pKa but varying size. Our results underscore the need for extensive and multifaceted variation to assess and minimize steric and positioning effects and determine effective molarities that estimate catalytic contributions. We obtained consensus effective molarities of ∼5 × 10(4) M for KSI from Comamonas testosteroni (tKSI) and ∼10(3) M for KSI from Pseudomonas putida (pKSI). An X-ray crystal structure of a tKSI general base mutant showed no additional structural rearrangements, and double mutant cycles revealed similar contributions from an oxyanion hole mutation in the wild-type and base-rescued reactions, providing no indication of mutational effects extending beyond the general base site. Thus, the high effective molarities suggest a large catalytic contribution associated with the general base. A significant portion of this effect presumably arises from positioning of the base, but its large magnitude suggests the involvement of additional catalytic mechanisms as well.
authors.affiliation: Department of Biochemistry, ‡Department of Chemistry, #Department of Chemical Engineering, §Stanford ChEM-H, Stanford University , Stanford, California 94305, United States.
authors.forename: Vandana
authors.initials: V
authors.lastname: Lamba
authors.affiliation: Department of Biochemistry, ‡Department of Chemistry, #Department of Chemical Engineering, §Stanford ChEM-H, Stanford University , Stanford, California 94305, United States.
authors.forename: Filip
authors.initials: F
authors.lastname: Yabukarski
authors.affiliation: Department of Biochemistry, ‡Department of Chemistry, #Department of Chemical Engineering, §Stanford ChEM-H, Stanford University , Stanford, California 94305, United States.
authors.forename: Margaux
authors.initials: M
authors.lastname: Pinney
authors.affiliation: Department of Biochemistry, ‡Department of Chemistry, #Department of Chemical Engineering, §Stanford ChEM-H, Stanford University , Stanford, California 94305, United States.
authors.forename: Daniel
authors.initials: D
authors.lastname: Herschlag
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D002244:Carbon / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D020604:Comamonas testosteroni / Q000201:enzymology; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007535:Isomerases / Q000378:metabolism; D007664:Ketosteroids / Q000737:chemistry*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016958:Pseudomonas putida / Q000201:enzymology; D013251:Steroid Isomerases / Q000737:chemistry*
chemical_list: D007664:Ketosteroids; D001224:Aspartic Acid; D002244:Carbon; D007535:Isomerases; D013251:Steroid Isomerases