Dissecting the paradoxical effects of hydrogen bond mutations in the ketosteroid isomerase oxyanion hole

Abstract

The catalytic importance of enzyme active-site interactions is frequently assessed by mutating specific residues and measuring the resulting rate reductions. This approach has been used in bacterial ketosteroid isomerase to probe the energetic importance of active-site hydrogen bonds donated to the dienolate reaction intermediate. The conservative Tyr16Phe mutation impairs catalysis by 10 5 -fold, far larger than the effects of hydrogen bond mutations in other enzymes. However, the less-conservative Tyr16Ser mutation, which also perturbs the Tyr16 hydrogen bond, results in a lesssevere 10 2 -fold rate reduction. To understand the paradoxical effects of these mutations and clarify the energetic importance of the Tyr16 hydrogen bond, we have determined the 1.6-Å resolution x-ray structure of the intermediate analogue, equilenin, bound to the Tyr16Ser mutant and measured the rate effects of mutating Tyr16 to Ser, Thr, Ala, and Gly. The nearly identical 200-fold rate reductions of these mutations, together with the 6.4-Å distance observed between the Ser16 hydroxyl and equilenin oxygens in the x-ray structure, strongly suggest that the more moderate rate effect of this mutant is not due to maintenance of a hydrogen bond from Ser at position 16. These results, additional spectroscopic observations, and prior structural studies suggest that the Tyr16Phe mutation results in unfavorable interactions with the dienolate intermediate beyond loss of a hydrogen bond, thereby exaggerating the apparent energetic benefit of the Tyr16 hydrogen bond relative to the solution reaction. These results underscore the complex energetics of hydrogen bonding interactions and sitedirected mutagenesis experiments.active-site environment | enzymatic catalysis | protein cavities | site-directed mutagenesis O ur knowledge of enzyme function has been greatly advanced by a combination of structure determination and sitedirected mutagenesis. These approaches have yielded detailed models of active-site architectures and identified the key catalytic groups positioned near bound substrates whose mutation results in large rate reductions. From this knowledge and an understanding of basic properties of chemical reactivity, detailed reaction mechanisms have been elucidated for numerous enzymes (1). Nevertheless, incisive understanding of the energetic contributions of physical interactions within active sites to the extraordinary 10 10 -10 20 -fold rate enhancements of enzymes remains a central challenge of biochemistry and a key hurdle in rational enzyme design (2-6).Site-directed mutagenesis has been used extensively in bacterial ketosteroid isomerase from Pseudomonas putida (pKSI) and Commamonas testosteroni (tKSI) to probe the catalytic importance of active-site hydrogen bonds. KSI catalyzes a reversible double-bond isomerization within steroid substrates that proceeds via a dienolate intermediate stabilized by hydrogen bonds donated by Tyr16 (pKSI numbering) and protonated Asp103 within an active-site oxyanion hole (Fig. 1A). The conservative Tyr...

Quantitative dissection of hydrogen bond-mediated proton transfer in the ketosteroid isomerase active site

“…Hydrogen bonds are typically probed in a coarse fashion by ablating them via site-directed mutagenesis and evaluating the functional consequence of their removal. Although this approach can highlight their general functional importance, it does not reveal the physical properties of the intact hydrogen bonds that underpin their functional roles (8)(9)(10)48), and the energetic effects of mutations can have as much to do with surrounding structural rearrangements in response to hydrogen bond ablation as they do with properties of the hydrogen bonds themselves (49)(50)(51)(52). In contrast to these common mutagenic approaches, we have leveraged favorable features of KSI to interrogate the physical and energetic properties of the intact hydrogen bond network formed in the KSI active site and to study the effects of internal charge rearrangement on electrostatic fields within the active site.…”

Use of anion–aromatic interactions to position the general base in the ketosteroid isomerase active site

“…Mutations were confirmed by sequencing miniprep DNA from DH5α cells. Proteins were expressed and purified as described previously (41).…”

Novel Approach for Identifying Key Residues in Enzymatic Reactions: Proton Abstraction in Ketosteroid Isomerase

“…Additionally, it is known that different mutations can give different effects on an enzymatic reaction; in the case of the KSI isomerization, the decrease in the rate constant caused by the Tyr14Phe mutation is about 2 orders of magnitude larger than the decreases caused by the Tyr14Ala and Tyr14Gly mutations. 18 It was suggested that substituting Tyr14 with smaller residues results in the formation of a water cavity that stabilizes the negative charge on the substrate oxygen via hydrogen bonds. 18 for each deletion to analyze.…”

“…18 It was suggested that substituting Tyr14 with smaller residues results in the formation of a water cavity that stabilizes the negative charge on the substrate oxygen via hydrogen bonds. 18 for each deletion to analyze. Thus, the computational time increases essentially linearly with the number of deletions.…”

“…proton transfer from a substrate to an Asp. The catalytic reaction and the active site structure of KSI have been studied extensively by experimental techniques [12][13][14][15][16][17][18][19][20][21] and by theoretical computations [22][23][24][25][26][27][28] . It has been shown that KSI works by a preorganized active site which electrostatically stabilizes the intermediate state and the transition states via hydrogen bonds to the substrate.…”