scholarly journals Acid–base catalysis in Leuconostoc mesenteroides sucrose phosphorylase probed by site-directed mutagenesis and detailed kinetic comparison of wild-type and Glu237→Gln mutant enzymes

2007 ◽  
Vol 403 (3) ◽  
pp. 441-449 ◽  
Author(s):  
Alexandra Schwarz ◽  
Lothar Brecker ◽  
Bernd Nidetzky
Keyword(s):  
Leuconostoc Mesenteroides ◽  
Ph Dependence ◽  
Base Catalysis ◽  
Glycoside Hydrolases ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Acid Base ◽  
Sucrose Phosphorylase ◽  
Nmr Studies ◽  
Enzymatic Function

The role of acid–base catalysis in the two-step enzymatic mechanism of α-retaining glucosyl transfer by Leuconostoc mesenteroides sucrose phosphorylase has been examined through site-directed replacement of the putative catalytic Glu237 and detailed comparison of purified wild-type and Glu237→Gln mutant enzymes using steady-state kinetics. Reactions with substrates requiring Brønsted catalytic assistance for glucosylation or deglucosylation were selectively slowed at the respective step, about 105-fold, in E237Q. Azide, acetate and formate but not halides restored catalytic activity up to 300-fold in E237Q under conditions in which the deglucosylation step was rate-determining, and promoted production of the corresponding α-glucosides. In situ proton NMR studies of the chemical rescue of E237Q by acetate and formate revealed that enzymatically formed α-glucose 1-esters decomposed spontaneously via acyl group migration and hydrolysis. Using pH profiles of kcat/Km, the pH dependences of kinetically isolated glucosylation and deglucosylation steps were analysed for wild-type and E237Q. Glucosylation of the wild-type proceeded optimally above and below apparent pKa values of about 5.6 and 7.2 respectively whereas deglucosylation was dependent on the apparent single ionization of a group of pKa≈5.8 that must be deprotonated for reaction. Glucosylation of E237Q was slowed below apparent pKa≈6.0 but had lost the high pH dependence of the wild-type. Deglucosylation of E237Q was pH-independent. The results allow unequivocal assignment of Glu237 as the catalytic acid–base of sucrose phosphorylase. They support a mechanism in which the pKa of Glu237 cycles between ≈7.2 in free enzyme and ≈5.8 in glucosyl enzyme intermediate, ensuring optimal participation of the glutamate residue side chain at each step in catalysis. Enzyme deglucosylation to an anionic nucleophile took place with Glu237 protonated or unprotonated. The results delineate how conserved active-site groups of retaining glycoside hydrolases can accommodate enzymatic function of a phosphorylase.

The roles of Tyr91 and Lys162 in general acid–base catalysis in the pigeon NADP+-dependent malic enzyme

2008 ◽  
Vol 411 (3) ◽  
pp. 467-473 ◽  
Author(s):  
Cheng-Chin Kuo ◽  
Kuan-Yu Lin ◽  
Yau-Jung Hsu ◽  
Shu-Yu Lin ◽  
Yu-Tsen Lin ◽  
...  
Keyword(s):  
Malic Enzyme ◽  
Kinetic Studies ◽  
Base Catalysis ◽  
Site Directed Mutagenesis ◽  
Decarboxylase Activity ◽  
Wild Type ◽  
Acid Base ◽  
Enzymatic Mechanism ◽  
General Acid ◽  
Steady State Kinetic

The role of general acid–base catalysis in the enzymatic mechanism of NADP+-dependent malic enzyme was examined by detailed steady-state kinetic studies through site-directed mutagenesis of the Tyr91 and Lys162 residues in the putative catalytic site of the enzyme. Y91F and K162A mutants showed approx. 200- and 27000-fold decreases in kcat values respectively, which could be partially recovered with ammonium chloride. Neither mutant had an effect on the partial dehydrogenase activity of the enzyme. However, both Y91F and K162A mutants caused decreases in the kcat values of the partial decarboxylase activity of the enzyme by approx. 14- and 3250-fold respectively. The pH-log(kcat) profile of K162A was found to be different from the bell-shaped profile pattern of wild-type enzyme as it lacked a basic pKa value. Oxaloacetate, in the presence of NADPH, can be converted by malic enzyme into L-malate by reduction and into enolpyruvate by decarboxylation activities. Compared with wild-type, the K162A mutant preferred oxaloacetate reduction to decarboxylation. These results are consistent with the function of Lys162 as a general acid that protonates the C-3 of enolpyruvate to form pyruvate. The Tyr91 residue could form a hydrogen bond with Lys162 to act as a catalytic dyad that contributes a proton to complete the enol–keto tautomerization.

scholarly journals Study of the role of the highly conserved residues Arg9 and Arg64 in the catalytic function of human N-acetyltransferases NAT1 and NAT2 by site-directed mutagenesis

1997 ◽  
Vol 323 (1) ◽  
pp. 207-215 ◽  
Author(s):  
Claudine DELOMÉNIE ◽  
Geoffrey H. GOODFELLOW ◽  
Rajagopal KRISHNAMOORTHY ◽  
Denis M. GRANT ◽  
Jean-Marie DUPRET
Keyword(s):  
Ph Dependence ◽  
Conformational Stability ◽  
Base Catalysis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Base Catalysts ◽  
General Base ◽  
Catalytic Function ◽  
Transition State Stabilization

The arylamine N-acetyltransferases (NATs) NAT1 and NAT2 are responsible for the biotransformation of many arylamine and hydroxylamine xenobiotics. It has been proposed that NATs may act through a cysteine-linked acetyl-enzyme intermediate in a general base catalysis involving a highly conserved arginine residue such as Arg64. To investigate this possibility, we used site-directed mutagenesis and expression of recombinant human NAT1 and NAT2 in Escherichia coli. Sequence comparison with NATs from other species indicated that Arg9 and Arg64 are the only invariant basic residues. Either mutation of the presumed catalytic Cys68 residue or the simultaneous mutation of Arg9 and Arg64 to Ala produced proteins with undetectable enzyme activity. NAT1 or NAT2 singly substituted at Arg9 or Arg64 with Ala, Met, Gln or Lys exhibited unaltered Km values for arylamine acceptor substrates, but a marked loss of activity and stability. Finally, double replacement of Arg9/Arg64 with lysine in NAT1 altered the Km for arylamine substrates (decreased by 8-14-fold) and for acetyl-CoA (elevated 5-fold), and modified the pH-dependence of activity. Thus, through their positively charged side chains, Arg9 and Arg64 seem to contribute to the conformational stability of NAT1 and NAT2 rather than acting as general base catalysts. Our results also support a mechanism in which Arg9 and Arg64 are involved in substrate binding and transition-state stabilization of NAT1.

Folding and catalysis of the hairpin ribozyme

2005 ◽  
Vol 33 (3) ◽  
pp. 461-465 ◽  
Author(s):  
T.J. Wilson ◽  
M. Nahas ◽  
T. Ha ◽  
D.M.J. Lilley
Keyword(s):  
Single Molecule ◽  
Ph Dependence ◽  
Active Form ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Base Catalysis ◽  
Hairpin Ribozyme ◽  
Acid Base ◽  
Physiological Conditions ◽  
Single Molecule Enzymology

The active form of the hairpin ribozyme is brought about by the interaction of two formally unpaired loops. In a natural molecule, these are present on two adjacent arms of a four-way junction. Although activity can be obtained in molecules lacking this junction, the junction is important in the promotion of the folded state of the ribozyme under physiological conditions, at a rate that is faster than the chemical reaction. Single-molecule fluorescence resonance energy transfer studies show that the junction introduces a discrete intermediate into the folding process, which repeatedly juxtaposes the two loops and thus promotes their docking. Using single-molecule enzymology, the cleavage and ligation rates have been measured directly. The pH dependence of the rates is consistent with a role for nucleobases acting in general acid–base catalysis.

Expression and Characterization of Clustered Charge-to-Alanine Mutants of Low Mr Single-Chain Urokinase-Type Plasminogen Activator

1994 ◽  
Vol 71 (01) ◽  
pp. 134-140 ◽  
Author(s):  
S Ueshima ◽  
P Holvoet ◽  
H R Lijnen ◽  
L Nelles ◽  
V Seghers ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Solvent Accessibility ◽  
Site Directed Mutagenesis ◽  
Clot Lysis ◽  
Wild Type ◽  
Single Chain ◽  
Charged Amino Acids ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Pharmacokinetic Properties

SummaryIn an effort to modify the fibrinolytic and/or pharmacokinetic properties of recombinant low M r single-chain urokinase-type plasminogen activator (rscu-PA-32k), mutants were prepared by site-directed mutagenesis of clusters of charged amino acids with the highest solvent accessibility. The following mutants of rscu-PA-32k were prepared: LUK-2 (Lys 212, Glu 213 and Asp 214 to Ala), LUK-3 (Lys 243 and Asp 244 to Ala), LUK-4 (Arg 262, Lys 264, Glu 265 and Arg 267 to Ala), LUK-5 (Lys 300, Glu 301 and Asp 305 to Ala) and LUK-6 (Arg 400, Lys 404, Glu 405 and Glu 406 to Ala).The rscu-PA 32k moictic3 were expressed in High Five Ttichoplasiani cells, and purified to humugciicily from the conditioned cell culture medium, with recoveries of 0.8 to 3.7 mg/1. The specific fibrinolytic activities (220,000 to 300,000 IU/mg), the rates of plasminogen activation by the single-chain moieties and the rates of conversion In lwo chain moieties by plasmin were comparable for mutant and wild-type rscu PA 32k moieties, with the exception of LUK-5 which was virtually inactive. Equi-effective lysis (50% in 2 h) of 60 pi 125I-fibrin labeled plasma clots submerged in 0.5 ml normal human plasma was obtained with 0.7 to 0.8 μg/ml of wild-type or mutant rscu-PA-3?.k, except with LUK-5 (no significant lysis with 16 pg/ml). Following bolus injection in hamsters, all rscu-PA-32k moieties had a comparably rapid plasma clearance (1.3 to 2.7 ml/min), as a result of a short initial half-life (1.4 to 2.5 min). In hamsters with pulmonary embolism, continuous intravenous infusion over 60 min at a dose of 1 mg/kg, resulted in 53 to 72% clot lysis with the mutants, but only 23% with LUK-5, as compared to 36% for wild-type rscu-PA-32k.These data indicate that clustered charge-to-alanine mutants of rscu-PA-32k, designed to eliminate charged regions with the highest solvent accessibility, do not have significantly improved functional, fibrinolytic or pharmacokinetic properties.

Mechanism of hydration and isomerisation of 4-ethylidene-2,6-di-tert-butyl-2,5-cyclohexadien-1-one. Kinetics, acid-base catalysis and solvent deuterium isotope effects

1979 ◽  
Vol 44 (1) ◽  
pp. 110-122 ◽  
Author(s):  
Jiří Velek ◽  
Bohumír Koutek ◽  
Milan Souček
Keyword(s):  
Aqueous Medium ◽  
Rate Equation ◽  
Kinetic Data ◽  
Isotope Effects ◽  
Base Catalysis ◽  
Quinone Methide ◽  
Reaction Products ◽  
Acid Base ◽  
Deuterium Isotope Effects ◽  
Hydroxybenzyl Alcohol

Competitive hydration and isomerisation of the quinone methide I at 25 °C in an aqueous medium in the region of pH 2.4-13.0 was studied spectrophotometrically. The only reaction products in the studied range of pH are 4-hydroxybenzyl alcohol (II) and 4-hydroxystyrene (III). The form of the overall rate equation corresponds to a general acid-base catalysis. The mechanism of both reactions for three markedly separated pH regions is discussed on the basis of kinetic data and solvent deuterium effect.

Pulse polarographic study of the behaviour of some o,o'-dihydroxyazo-compounds

1989 ◽  
Vol 54 (5) ◽  
pp. 1219-1226 ◽  
Author(s):  
Enric Casassas ◽  
Miquel Esteban ◽  
Santiago Alier
Keyword(s):  
Carboxylic Acid ◽  
Reaction Mechanisms ◽  
Sulphonic Acid ◽  
Base Catalysis ◽  
Polarographic Study ◽  
Acid Base ◽  
Eriochrome Black T ◽  
Naphthoic Acid ◽  
Calcon Carboxylic Acid

The reduction of several o,o'-dihydroxyazo-compounds is studied by means of pulse polarographic techniques (DPP, NPP and RPP). The compounds studied are the following: 2-(2'-hydroxyphenylazo)-phenol (o,o'-dihydroxyazobenzene), 1-(2'-hydroxy-1'-naphthylazo)-2-naphthol-4-sulphonic acid (calcon or Eriochrome Blue Black R), 1-(2'-hydroxy-4'-sulpho-1'-naphthylazo)-2-hydroxy-3-naphthoic acid (calcon carboxylic acid), and 1-(1'-hydroxy-2'-naphthylazo)-6-nitro-2-naphthol-4-sulphonic acid (Eriochrome Black T). Correlations between Ip and Epand experimental variables (pH, T, conc.) and instrumental parameters (dropping time, t, and pulse magnitude, ΔE) are established. Reaction mechanisms formerly proposed are discussed on the basis of the new obtained results, and the ranges are defined where adsorption and/or acid-base catalysis are operative.

scholarly journals Suberoylanilide hydroxamic acid enhances the radiosensitivity of lung cancer cells through acetylated wild-type and mutant p53-dependent modulation of mitochondrial apoptosis

2021 ◽  
Vol 49 (2) ◽  
pp. 030006052098154
Author(s):  
Kan Wu ◽  
Xueqin Chen ◽  
Xufeng Chen ◽  
Shirong Zhang ◽  
Yasi Xu ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cancer Cells ◽  
Hydroxamic Acid ◽  
Site Directed Mutagenesis ◽  
Lung Cancer Cells ◽  
Mutant P53 ◽  
Suberoylanilide Hydroxamic Acid ◽  
Mitochondrial Apoptosis ◽  
Wild Type ◽  
Clonogenic Cell

Objective Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, has shown potential as a candidate radiosensitizer for many types of cancers. This study aimed to explore the radiosensitization mechanism of SAHA in lung cancer cells. Methods Mutations in p53 were generated by site-directed mutagenesis using polymerase chain reaction. Transfection was performed to generate H1299 cells carrying wild-type or mutant p53. The radiosensitizing enhancement ratio was determined by clonogenic assays. Mitochondrial apoptosis was detected using JC-1 staining and flow cytometry analysis. Results Our results showed that SAHA induced radiosensitization in H1299 cells expressing wild-type p53, p53R175H or p53P223L, but this enhanced clonogenic cell death was not observed in parental H1299 (p53-null) cells or H1299 cells expressing p53 with K120R, A161T and V274R mutations. In SAHA-sensitized cells, mitochondrial apoptosis was induced following exposure to irradiation. Additionally, we observed that a secondary mutation at K120 (K120R) could eliminate p53-mediated radiosensitization and mitochondrial apoptosis. Conclusions The results of this study suggest that wild-type and specific mutant forms of p53 mediate SAHA-induced radiosensitization by regulating mitochondrial apoptosis, and the stabilization of K120 acetylation by SAHA is the molecular basis contributing to radiosensitization in lung cancer cells.

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