Mutation of essential catalytic residues in pig citrate synthase

Alter, Gerald M.; Casazza, Joseph P.; Wang, Zhi; Nemeth, Peter; Srere, Paul A.; Evans, Claudia T.

doi:10.1021/bi00485a003

Cited by 70 publications

(105 citation statements)

References 35 publications

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“…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: 87%

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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 Inmentioning

confidence: 52%

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

confidence: 87%

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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“…DNA sequencing was performed using an ALF automated sequencer, and the cyclosequencing kit and protocol were provided by Amersham Pharmacia Biotech. Ampicillin and isopropyl-␤-D-thiogalactoside were purchased from U.S. Biochemical Corp. [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]acetyl-CoA was purchased from Moravek Biochemicals (Brea, CA) and 3-chloro- [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]propionic acid from American Radiolabeled Chemicals (St. Louis, MO). All other reagents were purchased from Sigma, Aldrich, Amersham Pharmacia Biotech, or Bio-Rad.…”

Section: Methodsmentioning

confidence: 99%

“…Residual HOD resonance was suppressed by lower-power presaturation. 13 C decoupling in 2 was carried out using the GARP decoupling scheme. The spectra were obtained using a 1 H spectral width of 6800 Hz; 16,000 data points were collected.…”

Section: Methodsmentioning

confidence: 99%

“…The spectra were obtained using a 1 H spectral width of 6800 Hz; 16,000 data points were collected. 13 C NMR (proton-decoupled) experiments were performed using a Bruker AC-300 instrument operating at 75.469 MHz for 13 C. All spectra were recorded at 21°C and referenced to tetramethylsilane. A sweep width of 16,000 Hz was used, and 16,000 data points were collected.…”

Section: Methodsmentioning

confidence: 99%

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3-Hydroxy-3-methylglutaryl-CoA Synthase

Chun¹,

Vinarov²,

Zajíček³

et al. 2000

Journal of Biological Chemistry

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Replacement of 3-hydroxy-3-methylglutaryl-CoA synthase's glutamate 95 with alanine diminishes catalytic activity by over 5 orders of magnitude. The structural integrity of E95A enzyme is suggested by the observation that this protein contains a full complement of acylCoA binding sites, as indicated by binding studies using a spin-labeled acyl-CoA. Active site integrity is also demonstrated by 13 C NMR studies, which indicate that E95A forms an acetyl-S-enzyme reaction intermediate with the same distinctive spectroscopic characteristics measured using wild type enzyme. The initial reaction steps are not disrupted in E95A, which exhibits normal levels of Michaelis complex and acetyl-S-enzyme intermediate. Likewise, E95A is not impaired in catalysis of the terminal reaction step, as indicated by efficient catalysis of a hydrolysis partial reaction. Single turnover experiments indicate defective C-C bond formation. The mechanism-based inhibitor, 3-chloropropionyl-CoA, efficiently alkylates E95A. This is compatible with the presence of a functional general base, raising the possibility that Glu 95 functions as a general acid. Demonstration of a significant upfield shift for the methyl protons of HMG-CoA synthase's acetyl-S-enzyme reaction intermediate suggests a hydrophobic active site environment that could elevate the pK a of Glu 95 as required to support its function as a general acid.3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) 1 synthase catalyzes the committed step in ketogenic and cholesterogenic pathways. Early work (1, 2) on the purified enzyme identified reaction intermediates that indicate that catalysis of HMGCoA production involves a three-step process (Scheme 1).Recombinant forms of both the avian (3) and human (4) enzyme have been expressed in Escherichia coli and isolated at high levels of purity. The recombinant human enzyme has been used to demonstrate that HMG-CoA synthase is the target of a potent antisteroidogenic drug (4). Mutagenesis work on the recombinant avian enzyme (3) has confirmed that Cys 129 is required to form the acetyl-S-enzyme intermediate (Scheme 1), a hypothesis that was initially advanced on the basis of protein modification by a mechanism-based inhibitor (5, 6) as well as sequence analysis of a peptide that harbors the acetyl-S-Cys 129 adduct (7).2 Additionally, kinetic characterization of mutants in which His 264 has been replaced implicates that residue (8) in binding of the second substrate, acetoacetyl-CoA. The functions of other active site residues that participate directly in catalysis have not yet been firmly established.The sensitivity of HMG-CoA synthase activity to treatment with a carboxyl-directed modification reagent led to a preliminary observation (9), which implicated Glu 95 as a residue that is important to reaction chemistry. This report documents the crucial role of Glu 95 in catalysis and indicates which of the three steps in the reaction is compromised upon replacement of the Glu 95 carboxyl. Finally, potential functions for Glu 95 are considered, and tests ai...

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Acetyl-CoA enolization in citrate synthase: A quantum mechanical/molecular mechanical (QM/MM) study

Mulholland¹,

Richards

1997

Proteins

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Citrate synthase forms citrate by deprotonation of acetyl-CoA followed by nucleophilic attack of this substrate on oxaloacetate, and subsequent hydrolysis. The rapid reaction rate is puzzling because of the instability of the postulated nucleophilic intermediate, the enolate of acetyl-CoA. As alternatives, the enol of acetyl-CoA, or an enolic intermediate sharing a proton with His-274 in a "low-barrier" hydrogen bond have been suggested. Similar problems of intermediate instability have been noted in other enzymic carbon acid deprotonation reactions. Quantum mechanical/molecular mechanical calculations of the pathway of acetyl-CoA enolization within citrate synthase support the identification of Asp-375 as the catalytic base. His-274, the proposed general acid, is found to be neutral. The acetyl-CoA enolate is more stable at the active site than the enol, and is stabilized by hydrogen bonds from His-274 and a water molecule. The conditions for formation of a low-barrier hydrogen bond do not appear to be met, and the calculated hydrogen bond stabilization in the reaction is less than the gas-phase energy, due to interactions with Asp-375 at the active site. The enolate character of the intermediate is apparently necessary for the condensation reaction to proceed efficiently.

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Mutation of essential catalytic residues in pig citrate synthase

Cited by 70 publications

References 35 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

3-Hydroxy-3-methylglutaryl-CoA Synthase

Acetyl-CoA enolization in citrate synthase: A quantum mechanical/molecular mechanical (QM/MM) study

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