Determining the catalytic role of remote substrate binding interactions in ketosteroid isomerase

Abstract

O ver the past decades, visualization of enzyme threedimensional structures has revealed that active sites are located in crevices or pockets. Within these active sites are found coenzymes, cofactors, metal ions, and side chains that participate in a rich array of chemical reactions. Based on this information and decades of enzymology and bioorganic chemistry, reasonable chemical mechanisms using these groups can be posited for most enzymatic reactions. Nevertheless, we still cannot quantitatively account for the enormous enzymatic rate enhancements and exquisite specificities observed by enzymes.Over the past century, Polanyi, Haldane, Pauling, and Jencks recognized a key distinguishing feature between chemical reactions taking place in an enzyme's active site and the corresponding reaction taking place in solution (1-4). Even if the enzymatic and solution reactions use the same cofactors, metal ions, and functional groups, an enzyme can use noncovalent interactions between the enzyme and substrate to accelerate the reaction. Indeed, binding interactions between enzymes and substrates, both directly at the site of chemical transformation and with nonreacting portions of the substrate have been shown to contribute to catalysis (e.g., refs. 5-19). The mechanisms by which these noncovalent interactions can assist catalysis have been widely discussed, as briefly described in the following two paragraphs.In the simplest scenario an enzyme can use binding interactions to localize the substrate to the active site. Beyond simple localization, these binding interactions can correctly position the reactive portion of a substrate relative to active site functional groups and relative to other substrates (7, 20-25). Concomitant with substrate binding solvent is displaced and excluded from the active site and solvent exclusion may be important in shaping the electrostatic environment within the active site (26,27). Indeed, solvent exclusion by substrate binding has been suggested to be important for catalysis in numerous enzymes (e.g., refs. 26-32).Jencks and others realized that remote binding interactions can do more than provide for tight binding between substrate and enzyme. Reactions of bound substrates can be facilitated by use of so-called ''intrinsic binding energy'', which can pay for substrate desolvation, distortion, electrostatic destabilization, and entropy loss (3,7,33,34). The term intrinsic binding energy is not a molecular explanation for catalysis, but rather provides a conceptual framework for analyzing the energetics of enzymatic catalysis. In this scenario the maximum binding energy is not realized in the ground state, because aspects of the bound state, such as restricted positioning of substrates, are energetically unfavorable relative to the interactions and freedom of motion in aqueous solution. However, changes associated with achievement of the transition state, such as charge and geometric rearrangements and the formation of partial covalent bonds between positioned substrates, allow the binding ...

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

“…Our results with 5(10)-ESTare summarized in Fig. 2 and Table S2 and are identical, within error, to independently determined values we reported in a separate study (16). The Tyr16Phe mutation decreases k cat by 20,000-fold, corresponding to a 5.8 kcal∕mol reduction in catalysis.…”

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

“…Similar to the previously reported ∼10 4 -fold difference in K M for reaction of S full and S mini with wild-type pKSI (35), the K M value for reaction with S mini is ∼5,000-fold higher than for reaction with S full for wild-type tKSI, suggesting that remote binding interactions are important for tKSI catalysis of reaction of free S (Fig. 8C).…”

“…The k cat value of 17 s −1 for reaction of S mini with wild-type tKSI is within twofold of the k cat value for reaction with S full , suggesting that binding interactions with the distal steroid rings are not important for specific transition-state stabilization, as previously observed for wild-type pKSI (35). Similar to the previously reported ∼10 4 -fold difference in K M for reaction of S full and S mini with wild-type pKSI (35), the K M value for reaction with S mini is ∼5,000-fold higher than for reaction with S full for wild-type tKSI, suggesting that remote binding interactions are important for tKSI catalysis of reaction of free S (Fig.…”

“…All reagents were commercially available and of the highest purity (≥97%). All buffers were prepared with reagent-grade materials or better; 5(10)EST was from Steraloids; 3-cyclohexen-1-one was synthesized as described previously (35).…”

“…Reactions with 5(10)EST (S full ) and 3-cyclohexen-1-one (S mini ) were monitored as described previously (35). Reactions were conducted at 25°C in 10 mM potassium phosphate (pH 7.2), 1 mM sodium EDTA, 2 mM DTT with 2% (vol/vol) DMSO added as a cosolvent for substrate solubility.…”