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.

Assessment of enzyme active site positioning and tests of catalytic mechanisms through X-ray-derived conformational ensembles

“…When a unimolecular reaction is compared to a bimolecular reaction, the ratio of rate constants has units of molar and, in the simplest scenario, this value, referred to as the Effective Molarity (EM), represents how well aligned the unimolecular groups are for reaction ( Figure 10A) (Kirby, 1980;Page and Jencks, 1971). The EM of ~10 3 -10 5 M determined for the KSI general base, for KSI from two species, is considerably higher than typical for a positioned general base and suggested the possibility of an unusually high conformational restriction of D40 (Lamba et al, 2016).…”

“…Nevertheless, the observed highly restricted motion in particular dimensions raises the possibility that other enzymes may be able to utilize geometric discrimination despite their ensemble nature. We also evaluated the origin of the high effective molarity of the KSI general base of 10 3 -10 5 M (Lamba et al, 2016), revealing a paradox between the high efficiency general base catalysis and its conformational leeway. Analysis of the residues surrounding the catalytic groups allowed us to build and test models for the interplay of forces responsible for conformational restrictions and motions, information that may be needed to meet the ultimate challenge of the routine design of new, highly-efficient enzymes.…”

Electric Fields and Enzyme Catalysis

“…It is noteworthy that Lamba et al (116) used chemical rescue experiments to estimate the rate acceleration derived from the positioning of Asp40 (10 2 - to 10 3 -fold), which we had qualitatively predicted (150), because in combination with the contribution we assign to electric field catalysis (10 5 -fold) (118), the two now account for the totality of KSI’s rate enhancement ( k cat / k uncat = 10 7.5 ). These weights assigned to chemical positioning and electric field catalysis are also consistent with mutational studies that monitored the loss in catalysis following mutations that misposition the base or remove the oxyanion hole (117, 151, 161).…”

“…Numerous experiments have highlighted the importance of the general base, Asp40, and its precise positioning with respect to the steroid substrate, which constitutes an important element in KSI catalysis (116, 117). In addition, the active site provides an environment, sometimes called the oxyanion hole, that stabilizes the dienolate-like transition state via H-bonds (46) (Figure 4 a ).…”

Kemp Eliminase Activity of Ketosteroid Isomerase

“…33–35 To prevent contamination, the deoxycholate affinity column as well as FPLC loops and gel filteration column Superose 12 were washed 6 M guanidinium, 40 mM potassium phosphate, pH 7.2 buffer followed by 40 mM potassium phosphate, 1 mM EDTA, pH 7.2 buffer prior to each protein purification as described. 35 Phosphate buffer was removed via a Zeba Spin Desalting column (Thermo Fisher). Identity of the mutant was confirmed by mass spectrometry and purity of > 95% was determined using polyacrylamide SDS gel electrophoresis.…”