Evaluation of the Catalytic Contribution from a Positioned General Base in Ketosteroid Isomerase

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
× 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.