scholarly journals Contribution of five amino acid residues in the glutathione-binding site to the function of human glutathione transferase P1-1

1992 ◽  
Vol 285 (2) ◽  
pp. 377-381 ◽  
Author(s):  
M Widersten ◽  
R H Kolm ◽  
R Björnestedt ◽  
B Mannervik
Keyword(s):  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Catalytic Efficiency ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Ph Profile ◽  
Mutant Proteins ◽  
Catalytically Active ◽  
Active Site Cavity ◽  
Important Structure

Five amino acids in proximity to GSH bound in the active-site cavity of human Class Pi glutathione transferase (GST) P1-1 were mutated by oligonucleotide-directed site-specific mutagenesis. The following mutations gave catalytically active mutant proteins with the proper dimeric structure: Arg14----Ala, Lys45----Ala, Gln52----Ala, Gln65----His and Asp99----Asn. The mutation Gln65----Ala was also made, but the protein was not characterized because of its poor catalytic activity. Residues Arg14, Lys45, Gln52 and Gln65 all contribute to binding of glutathione, and the substitutions caused an approx. 10-fold decrease in affinity, corresponding to 5 kJ/mol, except for Arg14, for which the effect was larger. In addition, Arg14 appears to have an important structure role, since the Arg14----Ala mutant demonstrated a significantly lower stability as compared with the wild-type and the other mutant enzymes. Asp99 primarily contributes to catalysis rather than to binding. The kcat./Km-versus-pH profile for the Asp99----Asn mutant is shifted by 0.5 pH unit in the alkaline direction, and it is proposed that Asp99 may participate in proton transfer in the catalytic mechanism. The possibility of redesigning the substrate specificity for GSTs was shown by the fact that the mutant Lys45----Ala displayed a higher catalytic efficiency with GSH monoethyl ester than with its natural substrate, GSH.

scholarly journals Intragenic suppression among CDC34 (UBC3) mutations defines a class of ubiquitin-conjugating catalytic domains.

1995 ◽  
Vol 15 (10) ◽  
pp. 5635-5644 ◽  
Author(s):  
Y Liu ◽  
N Mathias ◽  
C N Steussy ◽  
M G Goebl
Keyword(s):  
Temperature Sensitive ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Cloning And Sequencing ◽  
Mutant Proteins ◽  
Catalytic Domains ◽  
Conserved Regions ◽  
E2 Enzymes ◽  
Key Residues

Ubiquitin-conjugating (E2) enzymes contain several regions within their catalytic domains that are highly conserved. However, within some of these conserved regions are several residues that may be used to define different classes of catalytic domains for the E2 enzymes. One class can be defined by the Ubc1 protein, which contains K-65, D-90, and D-120, while the corresponding positions within the Cdc34 (Ubc3) protein, which defines a second class of enzymes, contain S-73, S-97, and S-139, respectively. The presence of these differences within otherwise highly conserved regions of this family suggests that these residues may be critical for the specificity of Cdc34 function or regulation. Therefore, we have constructed a series of cdc34 alleles encoding mutant proteins in which these serine residues have been changed to other amino acid residues, including alanine and aspartic acid. In vivo complementation studies showed that S-97, which lies near the active site C-95, is essential for Cdc34 function. The addition of a second mutation in CDC34, which now encoded both the S97D and S73K changes, restored partial function to the Cdc34 enzyme. Moreover, the deletion of residues 103 to 114 within Cdc34, which are not present in the Ubc1-like E2s, allowed the S73K/S97D mutant to function as efficiently as wild-type Cdc34 protein. Finally, the cloning and sequencing of the temperature-sensitive alleles of CDC34 indicated that A-62 is also unique to the Cdc34 class of E2 enzymes and that mutations at this position can be detrimental to Cdc34 function. Our results suggest that several key residues within conserved regions of the E2 enzyme family genetically interact with each other and define a class of E2 catalytic domains.

scholarly journals The Role of Glutathione in the Isomerization of Δ5-Androstene- 3,17-dione Catalyzed by Human Glutathione Transferase A1-1

2001 ◽  
Vol 276 (15) ◽  
pp. 11698-11704 ◽  
Author(s):  
Pär L. Pettersson ◽  
Bengt Mannervik
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Ph Dependence ◽  
Enzymatic Catalysis ◽  
Catalytic Efficiency ◽  
Protonated Form ◽  
Hydroxyl Group ◽  
Isomerization Reaction

Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Δ5-androstene-3,17-dione (AD) into Δ4-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect.S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr9into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pKavalue of the enzyme-bound glutathione thiol. Thus, Tyr9promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr9residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3β-hydroxysteroid dehydrogenase.

scholarly journals On the chemical diversity of the prebiotic ocean of early Earth

2014 ◽  
Vol 14 (3) ◽  
pp. 497-504
Author(s):  
Carlo Canepa
Keyword(s):  
Amino Acid ◽  
Average Length ◽  
Chemical Species ◽  
Catalytic Efficiency ◽  
Chemical Diversity ◽  
Aqueous Environment ◽  
Amino Acid Residues ◽  
Catalytically Active ◽  
Order Of Magnitude ◽  
The Mean

AbstractThis work investigates the consequences on the diverse number of chemical species in a pre-biotic terrestrial aqueous environment endowed with an amino acid source induced by the spontaneous build-up of catalytically active polypeptides from amino acid monomers. The assumed probability that a randomly formed polypeptide exhibits catalytic properties is dependent on constraining both the chemical identity and the position of a fraction of the amino acid residues. Within this hypothesis, and using values of the average length n of the catalytic polypeptides about one half of the present-day enzymes, the stationary-state concentration of the catalytically active polypeptides is ≈10−30 −10−19 M, and the ratio of the concentration of a product of a catalytic process to the initial concentration of the corresponding substrate is predicted to be ≈10−6−105. Matching the mean life of each catalytic polypeptide to the mean life of its substrate (λ ≈ ω) is only possible by significantly raising the intensity of the source of the amino acid monomers. Under these hypothetical optimal conditions, the mean lives of the catalytic polypeptides and their substrates have values ω−1 ≈ λ−1 ≈10 yr and the asymptotic concentration of each product is of the same order of magnitude as the concentration of the substrate. In all cases the catalytic efficiency necessary to form the active peptides takes the typical values of present-day enzymes.

scholarly journals Catalytic mechanism of Zn2+-dependent polyol dehydrogenases: kinetic comparison of sheep liver sorbitol dehydrogenase with wild-type and Glu154→Cys forms of yeast xylitol dehydrogenase

2007 ◽  
Vol 404 (3) ◽  
pp. 421-429 ◽  
Author(s):  
Mario Klimacek ◽  
Heidemarie Hellmer ◽  
Bernd Nidetzky
Keyword(s):  
Rate Constant ◽  
Catalytic Mechanism ◽  
Isotope Effects ◽  
Bound Water ◽  
Cysteine Residue ◽  
Xylitol Dehydrogenase ◽  
Sorbitol Dehydrogenase ◽  
Wild Type ◽  
Ph Profile ◽  
Sheep Liver

Co-ordination of catalytic Zn2+ in sorbitol/xylitol dehydrogenases of the medium-chain dehydrogenase/reductase superfamily involves direct or water-mediated interactions from a glutamic acid residue, which substitutes a homologous cysteine ligand in alcohol dehydrogenases of the yeast and liver type. Glu154 of xylitol dehydrogenase from the yeast Galactocandida mastotermitis (termed GmXDH) was mutated to a cysteine residue (E154C) to revert this replacement. In spite of their variable Zn2+ content (0.10–0.40 atom/subunit), purified preparations of E154C exhibited a constant catalytic Zn2+ centre activity (kcat) of 1.19±0.03 s−1 and did not require exogenous Zn2+ for activity or stability. E154C retained 0.019±0.003% and 0.74±0.03% of wild-type catalytic efficiency (kcat/Ksorbitol=7800±700 M−1· s−1) and kcat (=161±4 s−1) for NAD+-dependent oxidation of sorbitol at 25 °C respectively. The pH profile of kcat/Ksorbitol for E154C decreased below an apparent pK of 9.1±0.3, reflecting a shift in pK by about +1.7–1.9 pH units compared with the corresponding pH profiles for GmXDH and sheep liver sorbitol dehydrogenase (termed slSDH). The difference in pK for profiles determined in 1H2O and 2H2O solvent was similar and unusually small for all three enzymes (≈+0.2 log units), suggesting that the observed pK in the binary enzyme–NAD+ complexes could be due to Zn2+-bound water. Under conditions eliminating their different pH-dependences, wild-type and mutant GmXDH displayed similar primary and solvent deuterium kinetic isotope effects of 1.7±0.2 (E154C, 1.7±0.1) and 1.9±0.3 (E154C, 2.4±0.2) on kcat/Ksorbitol respectively. Transient kinetic studies of NAD+ reduction and proton release during sorbitol oxidation by slSDH at pH 8.2 show that two protons are lost with a rate constant of 687±12 s−1 in the pre-steady state, which features a turnover of 0.9±0.1 enzyme equivalents as NADH was produced with a rate constant of 409±3 s−1. The results support an auxiliary participation of Glu154 in catalysis, and possible mechanisms of proton transfer in sorbitol/xylitol dehydrogenases are discussed.

The Specific Contribution of Amino Acids 334 and 335 from Factor Va Heavy Chain to the Catalytic Efficiency of Prothrombinase.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1027-1027
Author(s):  
Melissa A. Blum ◽  
Tivadar Orban ◽  
Daniel O. Beck ◽  
Michael Kalafatis
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Heavy Chain ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Factor Va ◽  
Cofactor Activity ◽  
Type Factor

Abstract The prothrombinase complex, composed of the enzyme factor Xa, the cofactor factor Va, and the substrate prothrombin associated on a cell surface in the presence of divalent metal ions, catalyzes the activation of prothrombin to thrombin 300,000-fold more effectively than the enzyme, factor Xa, alone. We have demonstrated that amino acids E323, Y324 and E330, V331 are binding sites for factor Xa on the factor Va heavy chain and are required for coordinating the spatial arrangement of enzyme and substrate directing prothrombin cleavage at two spatially distinct sites. We have also demonstrated that amino acid region 332–336 contains residues that are involved in cofactor function. Peptide studies have identified amino acid residues 334DY335 as major participants in factor Va cofactor activity. We have employed site-directed mutagenesis to study the effect of these amino acids on the catalytic efficiency of prothrombinase. Recombinant factor V molecules with the mutations D334K and Y335F, designated factor VKF, and D334A and Y335A, designated factor VAA were produced, transiently transfected, expressed in COS7L cells, and purified. Kinetic studies demonstrate that while factor VaKF has a KD for factor Xa similar to the KD observed for wild type factor Va, the kcat of prothrombinase assembled with factor VaKF has approximately a 1.5-fold decreased value compared to kcat of prothrombinase assembled with the wild type cofactor molecule. On the contrary, prothrombinase assembled with factor VaAA was found to have a nearly 10-fold decrease kcat, compared to prothrombinase assembled with wild type factor Va. This data suggest that not all amino acid substitutions are well tolerated at positions 334–335. Analysis of the sequence 323–340 using the recently published completed model of coagulation factor Va (pdb entry 1Y61) revealed that amino acids 334–335 are located at the end of a beta-sheet. To ascertain the importance of these mutants and their contribution to cofactor activity we have combined the mutations of amino acids 334–335 with mutations at amino acids 323–324 (E323F, Y324F) and 330–331 (E330M, V331I). We thus created quadruple mutants resulting in recombinant factor VFF/KF, factor VFF/AA, factor VMI/KF and factor VMI/AA. These molecules were transiently expressed in COS-7L cells and studied for their ability to be incorporated into prothrombinase. Free energies associated with the catalytic efficiencies of prothrombinase assembled with each mutant were also calculated (ΔΔGint). The ΔΔGint of interaction for the double mutants, factor VaFF/KF and factor VaMI/KF, had positive values indicating that the side chains of amino acids 330EV331, 323EY324 and 334DY335 located in and around the factor Xa binding site interact in a synergistic manner resulting in the destabilization of the transition state complex and a decelerated rate of catalysis. Conversely, combining the factor Xa binding site mutants with recombinant factor VaAA result in ΔΔGint values of approximately zero. In conclusion, the data demonstrate that replacement of amino acids 334–335 by two hydrophilic residues results in decreased cofactor function. In contrast, replacement of these amino acids by two small hydrophobic residues do not appear to be well tolerated by the cofactor resulting in severely impaired cofactor activity. Altogether, these data demonstrate the importance of amino acid residues D334 and Y335 for the rearrangement of enzyme and substrate required for efficient catalysis.

scholarly journals Mu-class glutathione transferase from Xenopus laevis: molecular cloning, expression and site-directed mutagenesis

2002 ◽  
Vol 365 (3) ◽  
pp. 685-691 ◽  
Author(s):  
Antonella De LUCA ◽  
Bartolo FAVALORO ◽  
Stefania ANGELUCCI ◽  
Paolo SACCHETTA ◽  
Carmine Di ILIO
Keyword(s):  
Xenopus Laevis ◽  
Catalytic Mechanism ◽  
Glutathione Transferase ◽  
Structural Instability ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Calculated Molecular Mass

A cDNA encoding a Mu-class glutathione transferase (XlGSTM1-1) has been isolated from a Xenopus laevis liver library, and its nucleotide sequence has been determined. XlGSTM1-1 is composed of 219 amino acid residues with a calculated molecular mass of 25359Da. Unlike many mammalian Mu-class GSTs, XlGSTM1-1 has a narrow spectrum of substrate specificity and it is also less effective in conjugating 1-chloro-2,4-dinitrobenzene. A notable structural feature of XlGSTM1-1 is the presence of the Cys-139 residue in place of the Glu-139, as well as the absence of the Cys-114 residue, present in other Mu-class GSTs, which is replaced by Ala. Site-directed mutagenesis experiments indicate that Cys-139 is not involved in the catalytic mechanism of XlGSTM1-1 but may be in part responsible for its structural instability, and experiments in vivo confirmed the role of this residue in stability. Evidence indicating that Arg-107 is essential for the 1-chloro-2,4-dinitrobenzene conjugation capacity of XlGSTM1-1 is also presented.

Structure of a class III engineered cephalosporin acylase: comparisons with class I acylase and implications for differences in substrate specificity and catalytic activity

2013 ◽  
Vol 451 (2) ◽  
pp. 217-226 ◽  
Author(s):  
Emily Golden ◽  
Rachel Paterson ◽  
Wan Jun Tie ◽  
Anandhi Anandan ◽  
Gavin Flematti ◽  
...  
Keyword(s):  
Transition State ◽  
Active Site ◽  
Catalytic Efficiency ◽  
Cephalosporin C ◽  
Class Iii ◽  
Wild Type ◽  
Serine Residue ◽  
Active Site Cavity ◽  
Β Chain ◽  
Enzyme Variant

The crystal structure of the wild-type form of glutaryl-7-ACA (7-aminocephalosporanic acid) acylase from Pseudomonas N176 and a double mutant of the protein (H57βS/H70βS) that displays enhanced catalytic efficiency on cephalosporin C over glutaryl-7-aminocephalosporanic acid has been determined. The structures show a heterodimer made up of an α-chain (229 residues) and a β-chain (543 residues) with a deep cavity, which constitutes the active site. Comparison of the wild-type and mutant structures provides insights into the molecular reasons for the observed enhanced specificity on cephalosporin C over glutaryl-7-aminocephalosporanic acid and offers the basis to evolve a further improved enzyme variant. The nucleophilic catalytic serine residue, Ser1β, is situated at the base of the active site cavity. The electron density reveals a ligand covalently bound to the catalytic serine residue, such that a tetrahedral adduct is formed. This is proposed to mimic the transition state of the enzyme for both the maturation step and the catalysis of the substrates. A view of the transition state configuration of the enzyme provides important insights into the mechanism of substrate binding and catalysis.

scholarly journals Site-directed mutagenesis establishes cysteine-110 as essential for enzyme activity in human γ-glutamyl hydrolase

1999 ◽  
Vol 343 (3) ◽  
pp. 551-555 ◽  
Author(s):  
Karen J. CHAVE ◽  
John GALIVAN ◽  
Thomas J. RYAN
Keyword(s):  
Enzyme Activity ◽  
Structural Changes ◽  
Catalytic Mechanism ◽  
Iodoacetic Acid ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Mutant Proteins ◽  
Key Enzyme

γ-Glutamyl hydrolase (GH), which hydrolyses the γ-glutamyl conjugates of folic acid, is a key enzyme in the maintenance of cellular folylpolyglutamate concentrations. The catalytic mechanism of GH is not known. Consistent with earlier reports that GH is sulphydryl-sensitive, we found that recombinant human GH is inhibited by iodoacetic acid, suggesting that at least one cysteine is important for activity [Rhee, Lindau-Shepard, Chave, Galivan and Ryan (1998) Mol. Pharmacol. 53, 1040-1046]. Using site-directed mutagenesis, the cDNA for human GH was altered to encode four different proteins each with one of four cysteine residues changed to alanine. Three of the mutant proteins had activities similar to wild-type GH and were inhibited by iodoacetic acid, whereas the C110A mutant had no activity. Cys-110 is conserved among the human, rat and mouse GH amino acid sequences. The wild-type protein and all four mutants had similar intrinsic fluorescence spectra, indicating no major structural changes had been introduced. These results indicate that Cys-110 is essential for enzyme activity and suggest that GH is a cysteine peptidase. These studies represent the first identification of the essential Cys residue in this enzyme and provide the beginning of a framework to determine the catalytic mechanism, important in defining GH as a therapeutic target.

scholarly journals Identification of Essential Amino Acid Residues of the NorM Na+/Multidrug Antiporter in Vibrio parahaemolyticus

2005 ◽  
Vol 187 (5) ◽  
pp. 1552-1558 ◽  
Author(s):  
Masato Otsuka ◽  
Makoto Yasuda ◽  
Yuji Morita ◽  
Chie Otsuka ◽  
Tomofusa Tsuchiya ◽  
...  
Keyword(s):  
Amino Acid ◽  
Ethidium Bromide ◽  
Vibrio Parahaemolyticus ◽  
Drug Transport ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Acidic Amino Acid ◽  
Wild Type ◽  
Transmembrane Region ◽  
Mutant Proteins

ABSTRACT NorM is a member of the multidrug and toxic compound extrusion (MATE) family and functions as a Na+/multidrug antiporter in Vibrio parahaemolyticus, although the underlying mechanism of the Na+/multidrug antiport is unknown. Acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM are conserved in one of the clusters of the MATE family. In this study, we investigated the role(s) of acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM by site-directed mutagenesis. Wild-type NorM and mutant proteins with amino acid replacements D32E (D32 to E), D32N, D32K, E251D, E251Q, D367A, D367E, D367N, and D367K were expressed and localized in the inner membrane of Escherichia coli KAM32 cells, while the mutant proteins with D32A, E251A, and E251K were not. Compared to cells with wild-type NorM, cells with the mutant NorM protein exhibited reduced resistance to kanamycin, norfloxacin, and ethidium bromide, but the NorM D367E mutant was more resistant to ethidium bromide. The NorM mutant D32E, D32N, D32K, D367A, and D367K cells lost the ability to extrude ethidium ions, which was Na+ dependent, and the ability to move Na+, which was evoked by ethidium bromide. Both E251D and D367N mutants decreased Na+-dependent extrusion of ethidium ions, but ethidium bromide-evoked movement of Na+ was retained. In contrast, D367E caused increased transport of ethidium ions and Na+. These results suggest that Asp32, Glu251, and Asp367 are involved in the Na+-dependent drug transport process.

scholarly journals Characterization of Dominantly Negative Mutant ClyA Cytotoxin Proteins in Escherichia coli

2003 ◽  
Vol 185 (18) ◽  
pp. 5491-5499 ◽  
Author(s):  
Sun Nyunt Wai ◽  
Marie Westermark ◽  
Jan Oscarsson ◽  
Jana Jass ◽  
Elke Maier ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Interactions ◽  
Single Channel ◽  
Dominant Negative ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Mutant Proteins ◽  
Negative Feature

ABSTRACT We report studies of the subcellular localization of the ClyA cytotoxic protein and of mutations causing defective translocation to the periplasm in Escherichia coli. The ability of ClyA to translocate to the periplasm was abolished in deletion mutants lacking the last 23 or 11 amino acid residues of the C-terminal region. A naturally occurring ClyA variant lacking four residues (183 to 186) in a hydrophobic subdomain was retained mainly in the cytosolic fraction. These mutant proteins displayed an inhibiting effect on the expression of the hemolytic phenotype of wild-type ClyA. Studies in vitro with purified mutant ClyA proteins revealed that they were defective in formation of pore assemblies and that their activity in hemolysis assays and in single-channel conductance tests was at least 10-fold lower than that of the wild-type ClyA. Tests with combinations of the purified proteins indicated that mutant and wild-type ClyA interacted and that formation of heteromeric assemblies affected the pore-forming activity of the wild-type protein. The observed protein-protein interactions were consistent with, and provided a molecular explanation for, the dominant negative feature of the mutant ClyA variants.

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