scholarly journals Site-directed mutagenesis of β-lactamase I. Single and double mutants of Glu-166 and Lys-73

1990 ◽  
Vol 272 (3) ◽  
pp. 613-619 ◽  
Author(s):  
R M Gibson ◽  
H Christensen ◽  
S G Waley
Keyword(s):  
Rate Constants ◽  
Double Mutant ◽  
Enzyme Mechanism ◽  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Burst Kinetics ◽  
Hydrolysis Of

Two single mutants and the corresponding double mutant of beta-lactamase I from Bacillus cereus 569/H were constructed and their kinetics investigated. The mutants have Lys-73 replaced by arginine (K73R), or Glu-166 replaced by aspartic acid (E166D), or both (K73R + E166D). All four rate constants in the acyl-enzyme mechanism were determined for the E166D mutant by the methods described by Christensen, Martin & Waley [(1990) Biochem. J. 266, 853-861]. Both the rate constants for acylation and deacylation for the hydrolysis of benzylpenicillin were decreased about 2000-fold in this mutant. In the K73R mutant, and in the double mutant, the rate constants for acylation were decreased about 100-fold and 10,000-fold respectively. All three mutants also had lowered values for the rate constants for the formation and dissociation of the non-covalent enzyme-substrate complex. The specificities of the mutants did not differ greatly from those of wild-type beta-lactamase, but the hydrolysis of cephalosporin C by the K73R mutant gave ‘burst’ kinetics.

scholarly journals Kinetic characterization of the acyl-enzyme mechanism for β-lactamase I

1988 ◽  
Vol 254 (3) ◽  
pp. 923-925 ◽  
Author(s):  
M T Martin ◽  
S G Waley
Keyword(s):  
Steady State ◽  
Rate Constants ◽  
Enzyme Mechanism ◽  
Beta Lactamase ◽  
Kinetic Characterization ◽  
Hydrolysis Of

beta-Lactamase I catalyses the hydrolysis of penicillins by an acyl-enzyme mechanism. A procedure was developed for determining the rate constants for the acylation and deacylation steps for the good substrates benzylpenicillin and phenoxymethylpenicillin; this depends on determining the fraction of enzyme that is present as acyl-enzyme in the steady state.

scholarly journals Rapid burst kinetics in the hydrolysis of 4-nitrophenyl acetate by penicillin G acylase from Kluyvera citrophila. Effects of mutation F360V on rate constants for acylation and de-acylation

1996 ◽  
Vol 316 (2) ◽  
pp. 409-412 ◽  
Author(s):  
A Roa ◽  
M L Goble ◽  
J L García ◽  
C Acebal ◽  
R Virden
Keyword(s):  
Rate Constants ◽  
Acetyl Derivative ◽  
Penicillin G Acylase ◽  
Penicillin G ◽  
Side Chain ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Burst Kinetics ◽  
Kinetics Of ◽  
Hydrolysis Of

The kinetics of release of 4-nitrophenol were followed by stopped-flow spectrophotometry with two 4-nitrophenyl ester substrates of penicillin G acylase from Kluyvera citrophila. With the ester of acetic acid, but not of propionic acid, there was a pre-steady-state exponential phase, the kinetics of which were inhibited by phenylacetic acid (a product of hydrolysis of specific substrates) to the extent predicted from Ki values. This was interpreted as deriving from rapid formation (73 mM-1·s-1) and slow hydrolysis (0.76 s-1) of an acetyl derivative of the side chain of the catalytic-centre residue Ser-290. With the mutant F360V, which differs from the wild-type enzyme in its ability to hydrolyse adipyl-L-leucine and has a kcat for 4-nitrophenyl acetate one-twentieth that of the wild-type enzyme, the corresponding values for the rates of formation and hydrolysis of the acetyl-enzyme were 11.1 mM-1·s-1 and 0.051 s-1 respectively. The ratio of these rate constants was three times that for the wild-type enzyme, suggesting that the mutant is less impaired in the rate of formation of an acetyl-enzyme than in its subsequent hydrolysis.

scholarly journals Site-directed mutagenesis and substrate-induced inactivation of β-lactamase I

1992 ◽  
Vol 288 (3) ◽  
pp. 1045-1051 ◽  
Author(s):  
S J Thornewell ◽  
S G Waley
Keyword(s):  
Alpha Helix ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Extracellular Medium ◽  
The Stability ◽  
Hydrolysis Of ◽  
Reduced Activity

The substrate-induced inactivation of beta-lactamase I from Bacillus cereus 569/H has been studied. Both the wild-type enzyme and mutants have been used. The kinetics follow a branched pathway of the type recently analysed [Waley (1991) Biochem. J. 279, 87-94]. The substrate cloxacillin (a penicillin) formed an acyl-enzyme (characterized by m.s.), and it was probably the instability of this intermediate that brought about inactivation. A disulphide bond was introduced into beta-lactamase I (the wild-type enzyme lacks this bond) by site-directed mutagenesis: Ala-77 and Ala-123 were replaced by cysteine. Spontaneous oxidation yielded the disulphide. The activity of this newly cross-linked enzyme was a little diminished, but the stability towards inactivation by cloxacillin was not increased. A second mutant of beta-lactamase I was studied: this mutant lacked the first 17 residues, i.e. the first alpha-helix. The mutant had reduced activity towards ordinary (non-inactivating) substrates and no hydrolysis of cloxacillin could be detected. These mutant enzymes were expressed in Bacillus subtilis, and were purified from the extracellular medium.

scholarly journals Active-site serine mutants of the Streptomyces albus G β-lactamase

1991 ◽  
Vol 277 (3) ◽  
pp. 647-652 ◽  
Author(s):  
F Jacob ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Ph Values ◽  
Streptomyces Albus ◽  
Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

Effect of Modifiers on the Hydrolysis of Basic and Neutral Peptides by Carboxypeptidase B

1975 ◽  
Vol 53 (7) ◽  
pp. 747-757 ◽  
Author(s):  
Graham J. Moore ◽  
N. Leo Benoiton
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Kinetic Behavior ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Carboxypeptidase B ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Basic Peptides ◽  
Hydrolysis Of

The initial rates of hydrolysis of Bz-Gly-Lys and Bz-Gly-Phe by carboxypeptidase B (CPB) are increased in the presence of the modifiers β-phenylpropionic acid, cyclohexanol, Bz-Gly, and Bz-Gly-Gly. The hydrolysis of the tripeptide Bz-Gly-Gly-Phe is also activated by Bz-Gly and Bz-Gly-Gly, but none of these modifiers activate the hydrolysis of Bz-Gly-Gly-Lys, Z-Leu-Ala-Phe, or Bz-Gly-phenyllactic acid by CPB. All modifiers except cyclohexanol display inhibitory modes of binding when present in high concentration.Examination of Lineweaver–Burk plots in the presence of fixed concentrations of Bz-Gly has shown that activation of the hydrolysis of neutral and basic peptides by CPB, as reflected in the values of the extrapolated parameters, Km(app) and keat, occurs by different mechanisms. For Bz-Gly-Gly-Phe, activation occurs because the enzyme–modifier complex has a higher affinity than the free enzyme for the substrate, whereas activation of the hydrolysis of Bz-Gly-Lys derives from an increase in the rate of breakdown of the enzyme–substrate complex to give products.Cyclohexanol differs from Bz-Gly and Bz-Gly-Gly in that it displays no inhibitory mode of binding with any of the substrates examined, activates only the hydrolysis of dipeptides by CPB, and has a greater effect on the hydrolysis of the basic dipeptide than on the neutral dipeptide. Moreover, when Bz-Gly-Lys is the substrate, cyclohexanol activates its hydrolysis by CPB by increasing both the enzyme–substrate binding affinity and the rate of the catalytic step, an effect different from that observed when Bz-Gly is the modifier.The anomalous kinetic behavior of CPB is remarkably similar to that of carboxypeptidase A, and is a good indication that both enzymes have very similar structures in and around their respective active sites. A binding site for activator molecules down the cleft of the active site is proposed for CPB to explain the observed kinetic behavior.

scholarly journals Mutant Firefly Luciferase Enzymes Resistant to the Inhibition by Sodium Chloride

2021 ◽  
Author(s):  
Satoshi Yawata ◽  
Kenichi Noda ◽  
Ai Shimomura ◽  
Akio Kuroda
Keyword(s):  
Sodium Chloride ◽  
Double Mutant ◽  
Luciferase Activity ◽  
Firefly Luciferase ◽  
Activity Level ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Original Activity ◽  
Double Mutation

Abstract ObjectivesFirefly luciferase, one of the most extensively studied enzymes, has numerous applications. However, luciferase activity is inhibited by sodium chloride. This study aims to expand the applications of firefly luciferase in the presence of sodium chloride.ResultsWe first obtained two mutant luciferase enzymes whose inhibition were alleviated and identified these mutations as Val288Ile and Glu488Val. Under dialysis condition (140 mM sodium chloride), the wild type was inhibited to 44% of its original activity level. In contrast, the single mutants, Val288Ile and Glu488Val, retained 67% and 79% of their original activity, respectively. Next, we introduced Val288Ile and Glu488Val mutations into the wild-type luciferase to create a double mutant using site-directed mutagenesis. Notably, the double mutant retained its activity more than 95% of that in the absence of sodium chloride.ConclusionsThe mutant luciferase, named luciferase CR, was found to retain its activity in various concentrations of sodium chloride. The inhibition of luciferase CR under dialysis condition was more alleviated than either Val288Ile or Glu488Val alone, suggesting that the effect of the double mutation was cumulative. We discussed the effect of mutations on the alleviation of the inhibition by sodium chloride.

Human cholinesterases

2020 ◽  
pp. 63-120
Author(s):  
Sergey Varfolomeev ◽  
Bella Grigorenko ◽  
Sofya Lushchekina ◽  
Alexander Nemuchin
Keyword(s):  
Active Site ◽  
The Body ◽  
Polymorphic Modification ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Mechanism Of Hydrolysis ◽  
The Central Nervous System ◽  
The Stability ◽  
Hydrolysis Of ◽  
Enzyme Reactivity

The work is devoted to modeling the elementary stages of the hydrolysis reaction in the active site of enzymes belonging to the class of cholinesterases — acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The study allowed to describe at the molecular level the effect of the polymorphic modification of BChE, causing serious physiolog ical consequences. Cholinesterase plays a crucial role in the human body. AChE is one of the key enzymes of the central nervous system, and BChE performs protective functions in the body. According to the results of calculations using the combined method of quantum and molecular mechanics (KM/MM), the mechanism of the hydrolysis of the native acetylcholine substrate in the AChE active center was detailed. For a series of ester substrates, a method for estimation of dependence of the enzyme reactivity on the structure of the substrate has been developed. The mechanism of hydrolysis of the muscle relaxant of succininylcholine BChE and the effect of the Asp70Gly polymorph on it were studied. Using various computer simulation methods, the stability of the enzyme-substrate complex of two enzyme variants with succinylcholine was studied.

scholarly journals Role of Sulfated Tyrosines of Thyroglobulin in Thyroid Hormonosynthesis

Endocrinology ◽  
2005 ◽  
Vol 146 (11) ◽  
pp. 4834-4843 ◽  
Author(s):  
Marie-Christine Nlend ◽  
David M. Cauvi ◽  
Nicole Venot ◽  
Odile Chabaud
Keyword(s):  
Amino Acid ◽  
Coupling Reaction ◽  
Short Life ◽  
Hormone Synthesis ◽  
Amino Acid Peptide ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Saturation Binding ◽  
Hydrolysis Of

Our previous studies showed that sulfated tyrosines (Tyr-S) are involved in thyroid hormone synthesis and that Tyr5, the main hormonogenic site of thyroglobulin (Tg), is sulfated. In the present paper, we studied the role of Tyr-S in the formation and activity of the peroxidase-Tg complex. Results show that noniodinated 35SO3-Tg specifically binds (Kd = 1.758 μm) to immobilized lactoperoxidase (LPO) via Tyr-S linkage by using saturation binding and competition experiments. We found that NIFEY-S, a 15-amino acid peptide corresponding to the NH2-end sequence of Tg and containing the hormonogenic acceptor Tyr5-S, was a better competitor than cholecystokinin and Tyr-S. 35SO3-Tg, iodinated without peroxidase, bound to LPO with a Kd (1.668 μm) similar to that of noniodinated Tg, suggesting that 1) its binding occurs via Tyr-S linkage and 2) Tyr-S requires peroxidase to be iodinated, whereas nonsulfated Tyr does not. Iodination of NIFEY-S with [125I]iodide showed that Tyr5-S iodination increased with LPO concentration, whereas iodination of a nonsulfated peptide containing the donor Tyr130 was barely dependent on LPO concentration. Enzymatic hydrolysis of iodinated Tg or NIFEY-S showed that the amounts of sulfated iodotyrosines also depended on LPO amount. Sulfated iodotyrosines were detectable in the enzyme-substrate complex, suggesting they have a short life before the coupling reaction occurs. Our data suggest that after Tyr-S binding to peroxidase where it is iodinated, the sulfate group is removed, releasing an iodophenoxy anion available for coupling with an iodotyrosine donor.

Sign in / Sign up

Export Citation Format

Share Document