Roles of Active Site Aromatic Residues in Catalysis by Ketosteroid Isomerase from <i>Pseudomonas putida </i>Biotype B

Kim, Do-Hyung; Nam, Gyu Hyun; Jang, Dogeun; Choi, Gildon; Joo, Sung Hyun; Kim, Jeong Sun; Oh, Byung Ha; Choi, Kwan Yong

doi:10.1021/bi991040m

Cited by 18 publications

(22 citation statements)

References 36 publications

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“…The rate decrease for the Phe54Ala mutation is similar to the 10-fold rate reduction that Choi and coworkers reported previously for Phe56Ala in the related Pseudomonas putida KSI (pKSI) for reaction of this substrate (18). In pKSI, Trp120 replaces Phe116, and the Trp120Ala mutation led to a 68-fold rate decrease (18).…”

Section: Resultssupporting

confidence: 86%

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

Schwans¹,

Sunden²,

Lassila³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Although the cation-pi pair, formed between a side chain or substrate cation and the negative electrostatic potential of a pi system on the face of an aromatic ring, has been widely discussed and has been shown to be important in protein structure and protein-ligand interactions, there has been little discussion of the potential structural and functional importance in proteins of the related anion-aromatic pair (i.e., interaction of a negatively charged group with the positive electrostatic potential on the ring edge of an aromatic group). We posited, based on prior structural information, that anion-aromatic interactions between the anionic Asp general base and Phe54 and Phe116 might be used instead of a hydrogen-bond network to position the general base in the active site of ketosteroid isomerase from Comamonas testosteroni as there are no neighboring hydrogenbonding groups. We have tested the role of the Phe residues using site-directed mutagenesis, double-mutant cycles, and high-resolution X-ray crystallography. These results indicate a catalytic role of these Phe residues. Extensive analysis of the Protein Data Bank provides strong support for a catalytic role of these and other Phe residues in providing anion-aromatic interactions that position anionic general bases within enzyme active sites. Our results further reveal a potential selective advantage of Phe in certain situations, relative to more traditional hydrogen-bonding groups, because it can simultaneously aid in the binding of hydrophobic substrates and positioning of a neighboring general base.enzyme catalysis | general-base catalysis | noncovalent interactions E nzymes use the same functional groups to achieve "chemical catalysis" as small-molecule catalysts, and yet enzymes attain much greater rate enhancements. A distinguishing feature of enzymes is that their reactions occur in highly specialized active sites that use noncovalent interactions to precisely position enzymatic functional groups relative to substrates and relative to other active-site features. Understanding how these groups are positioned within enzyme active sites is key for understanding the differences between enzymes and small-molecule catalysts.It is generally recognized that hydrophobic interactions can help bind and position substrates in enzyme active sites (1, 2). Beyond hydrophobic interactions, much attention has focused on the importance of hydrogen bonds in precisely positioning active-site groups directly involved in the chemical reaction (e.g., the catalytic triad in serine proteases), and the importance of hydrogen-bonding groups is supported by mutagenesis in many cases (e.g., refs. 1 and 3-5).In addition to hydrogen bonds, the cation-pi pair, formed between a side-chain or substrate cation and the negative electrostatic potential associated with the face of a pi system, has been widely discussed in hundreds of literature reports and has been shown to be important in both protein structure and proteinligand interactions (e.g., refs. 6 and 7). There has been much ...

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Section: Resultssupporting

confidence: 86%

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

Schwans¹,

Sunden²,

Lassila³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…The mutations reduced k cat /K M and k cat by up to Ϸ70,000-fold and by 17,000-fold, respectively, for S full and gave values in agreement with those from the literature (Table 1) (51). For each oxyanion hole mutant, the reductions in k cat /K M and k cat for reaction with S mini were within 3-fold of the respective decrease in k cat /K M or k cat for reaction with S full (Table 1).…”

Section: Effect Of Ksi Oxyanion Hole Mutations On Reaction Of the Sinsupporting

confidence: 88%

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

Schwans

Kraut

Herschlag

2009

Proc. Natl. Acad. Sci. U.S.A.

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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 ...

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“…Wild-type p KSI binds the substituted naphthols more weakly than p KSI D40N, and this weaker binding significantly limits our ability to probe the active site with multiple substituted naphthols as most bind with K d greater than 1 mM. However, equilenin binds to wild-type p KSI with a 1.8 µM K d (32), which is sufficient to assure nearly 100% binding under the conditions of the experiment. Based only on the contributions from the protonated and deprotonated forms of equilenin, the proton affinity of the oxyanion hole of wild-type p KSI is observed to match a solution p K a of 10.05 ± 0.03.…”

Section: Resultsmentioning

confidence: 99%

Proton Affinity of the Oxyanion Hole in the Active Site of Ketosteroid Isomerase

Childs

Boxer

2010

Biochemistry

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The absorption spectra of a series of inhibitors bound at the active site of Δ5-3-ketosteroid isomerase from Pseudomonas putida were found to exhibit substantial variations in the contributions of the protonated and deprotonated forms. Systematic variation of the inhibitor solution pKa combined with a method of quantifying the contributions of each protonation state showed the oxyanion hole in the active site of wild-type Δ5-3-ketosteroid isomerase to have a proton affinity equal to a solution pKa of 10.05 ± 0.03, which is similar to the measured pKa, 10.0, of the reaction intermediate. This observation supports the prediction of Cleland, Kreevoy, Frey, Gassman, and Gerlt that an enzyme utilizing a strong hydrogen bond for catalysis matches the proton affinity of the protein to the intermediate [Cleland, W.W. and Kreevoy, M.M. (1994) Science 264, 1887-189.], [Frey, P.A., Whitt, S., and Tobin, J. (1994) Science 264, 1927–1930], [Gerlt, J.A. and Gassman, P.G. (1993) Biochemistry 32, 11934 – 11952]. As the difference in proton affinity decreases, the strength of the hydrogen bond increases, and the closely matched proton affinity between the active site and the reaction intermediate supports the possibility that a short, strong hydrogen bond is catalytically relevant in Δ5-3-ketosteroid isomerase.

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Roles of Active Site Aromatic Residues in Catalysis by Ketosteroid Isomerase from Pseudomonas putida Biotype B

Cited by 18 publications

References 36 publications

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

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

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

Proton Affinity of the Oxyanion Hole in the Active Site of Ketosteroid Isomerase

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