5dre | pdb_00005dre
Crystal Structure of Ketosteroid Isomerase D38GP39GD99N mutant from Pseudomonas Testosteroni (tKSI)
Structural highlights
FunctionPublication Abstract from PubMedProton 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 multi-faceted variation to assess and minimize steric and positioning effects and determine effective molarities that estimate catalytic contributions. We obtained consensus effective molarities of ~5 X 104 M for KSI from Comamonas testosteroni (tKSI) and ~103 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. Evaluation of the Catalytic Contribution from a Positioned General Base in Ketosteroid Isomerase.,Lamba V, Yabukarski F, Pinney M, Herschlag D J Am Chem Soc. 2016 Jul 13. PMID:27410422[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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