Reaction Energetics of a Mutant 3-Oxo-.DELTA.5-steroid Isomerase with an Altered Active Site Base (D38E)

Zawrotny, Michael E.; Pollack, Ralph M.

doi:10.1021/bi00250a044

Cited by 16 publications

(31 citation statements)

References 26 publications

(41 reference statements)

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“…The Asp38Glu mutation led to a decrease of 240-fold for k cat (Fig. 6A), similar to the 300-fold decrease reported previously with a different KSI substrate (5-androstene-3,17-dione) (20). Whereas the Phe54Ala and Phe116Ala mutants reduce k cat Fig.…”

Section: Double-mutant Cycles To Test the Role Of Phe54 And Phe116 Insupporting

confidence: 87%

“…Such mutations are typically highly deleterious and have been shown to displace the position of the carboxylate by ∼1 Å relative to its wild-type position in several cases (19)(20)(21)(22)(23), a displacement similar to that observed for the Phe54Gly mutation described above.…”

Section: Double-mutant Cycles To Test the Role Of Phe54 And Phe116 Inmentioning

confidence: 52%

“…Prior studies have used mutations that add or remove a sidechain methylene group to test the importance of positioning active-site carboxylate groups (19)(20)(21)(22)(23)(24). Such mutations are typically highly deleterious and have been shown to displace the position of the carboxylate by ∼1 Å relative to its wild-type position in several cases (19)(20)(21)(22)(23), a displacement similar to that observed for the Phe54Gly mutation described above.…”

Section: Double-mutant Cycles To Test the Role Of Phe54 And Phe116 Inmentioning

confidence: 86%

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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: Double-mutant Cycles To Test the Role Of Phe54 And Phe116 Insupporting

confidence: 87%

Section: Double-mutant Cycles To Test the Role Of Phe54 And Phe116 Inmentioning

confidence: 52%

Section: Double-mutant Cycles To Test the Role Of Phe54 And Phe116 Inmentioning

confidence: 86%

See 1 more Smart Citation

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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“…Based on the sequence alignment, Asp-401 is the putative general acid/base in the WPD motif in bPTP␣-D1 whereas Glu-690 is the putative general acid/base in the WPE motif in bPTP␣-D2. It is found that replacement of Asp with Glu or vice versa generally leads to a reduction of up to 3 orders of magnitude in catalytic efficiency in enzymes that require a carboxylate group as a general acid/base (43)(44)(45)(46). Since an Asp is always found to be the general acid/base in the catalytic domains of cytoplasmic PTPases and the D1 domains of receptorlike PTPases, and since the D2 domains of receptor-like PTPases have much reduced catalytic activity, we asked the following question: can substitution of Glu-690 by an Asp in bPTP␣-D2 increase its catalytic activity to a level comparable to bPTP␣-D1?…”

Section: Kinetic Parameters Of Various Forms Of Rptp␣ Using Pnpp and mentioning

confidence: 99%

“…We illustrate that mutation of Asp-401 in the WPD motif of bPTP␣-D1 to an Ala decreases the k cat by 900-fold, confirming the importance of general acid/base in the D1 domain of receptor PTPases. Replacement of Asp with Glu or vice versa can have dramatic effects in enzymes that require a carboxylate group as a general acid/base (43)(44)(45)(46). Interestingly, the restoration of the general acid/base to an Asp at position 690 in bPTP␣-D2 only raises the activity by 4-fold.…”

Section: Kinetic Parameters Of Various Forms Of Rptp␣ Using Pnpp and mentioning

confidence: 99%

Comparative Kinetic Analysis and Substrate Specificity of the Tandem Catalytic Domains of the Receptor-like Protein-tyrosine Phosphatase α

Buist

Hertog

et al. 1997

Journal of Biological Chemistry

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The catalytic activity and substrate specificity of protein-tyrosine phosphatase ␣ (PTP␣) is primarily controlled by the membrane proximal catalytic domain (D1). The membrane distal (D2) domain of PTP␣ by itself is a genuine PTPase, possessing catalytic activity comparable to that of D1 using aryl phosphates as substrates. Surprisingly, k cat and k cat /K m for the D2-catalyzed hydrolysis of phosphotyrosine-containing peptides are several orders of magnitude reduced in comparison with those of D1. Substitution of the putative general acid/base Glu-690 in D2 by an Asp, which is invariably found in the WPD motifs in all cytoplasmic PTPases and all the D1 domains of receptor-like PTPases, only increases the k cat for D2 by 4-fold. Thus the much reduced D2 activity toward peptide substrates may be due to structural differences in the active sites other than the general acid/base. Alternatively, the D2 domain may have a functional active site with a highly stringent substrate specificity. PTP␣ display modest peptide substrate selectivity and are sensitive to charges adjacent to phosphotyrosine. In the sequence context of DADEpYLIPQQG (where pY stands for phosphotyrosine), the minimal sizes recognized by PTP␣ are either ADEpYLI or DADEpY-NH 2 . Protein-tyrosine phosphatases (PTPases)1 catalyze the hydrolysis of phosphoryl groups on tyrosine residues in proteins that are introduced by protein-tyrosine kinases. A tightly balanced phosphatase and kinase activity is required for proper cellular functions (1, 2). PTPases constitute a growing family (Ͼ70 members) of enzymes that can be structurally categorized into two major groups: receptor-like (transmembrane) and nonreceptor (intracellular) PTPases (3). Although many PTPases are proteins of greater than 400 amino acids, their catalytic domains are usually contained within a span of 250 residues referred to as the PTPase domain. This domain is the only structural element that has amino acid sequence identity among all PTPases from bacteria to mammals (4). The hallmark that defines the PTPase family is the active site sequence (H/V)C(X) 5 R(S/T), called the PTPase signature motif in the catalytic domain (5, 6). The receptor-like PTPases, exemplified by the leukocyte phosphatase CD45, generally have an extracellular domain, a single transmembrane region, and cytoplasmic PTPase domain(s). The intracellular PTPases, exemplified by PTP1B, contain a single catalytic domain and various amino-or carboxyl-terminal extensions including SH2 domains that may have targeting or regulatory functions.The cytoplasmic segment of many receptor-like PTPases have two tandem PTPase domains: D1, which is proximal to the membrane, and D2, which is distal to the membrane. The existence of homologous PTPase domains in the receptor-like PTPases raises the interesting possibility of differential functions or regulations of the two domains. However, the significance of the repeated PTPase domain in the receptor-like PTPases is not clear. One important question is whether both PTPase domains in the re...

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Keto–Enol Tautomerism in Enzymatic Reactions

Whitman

1999

Comprehensive Natural Products Chemistry

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Reaction Energetics of a Mutant 3-Oxo-.DELTA.5-steroid Isomerase with an Altered Active Site Base (D38E)

Cited by 16 publications

References 26 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

Comparative Kinetic Analysis and Substrate Specificity of the Tandem Catalytic Domains of the Receptor-like Protein-tyrosine Phosphatase α

Keto–Enol Tautomerism in Enzymatic Reactions

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