scholarly journals A dramatic change in the rate-limiting step of β-lactam hydrolysis results from the substitution of the active-site serine residue by a cysteine in the class-C β-lactamase of Enterobacter cloacae 908R

1993 ◽  
Vol 292 (2) ◽  
pp. 537-543 ◽  
Author(s):  
A Dubus ◽  
D Monnaie ◽  
C Jacobs ◽  
S Normark ◽  
J M FrÉre
Keyword(s):  
Active Site ◽  
Enterobacter Cloacae ◽  
Order Rate Constant ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Serine Residue ◽  
Rate Limiting Step ◽  
Class C ◽  
Rate Limiting ◽  
Selective Influence

A cysteine residue has been substituted for the active-site serine of the class-C beta-lactamase produced by Enterobacter cloacae 908R by site-directed mutagenesis. The modified protein exhibited drastically reduced kcat./Km values on all tested substrates. However, this decrease was due to increased Km values with some substrates and to decreased kcat. values with others. These apparently contradictory results could be explained by a selective influence of the mutation on the first-order rate constant characteristic of the acylation step, a hypothesis which was confirmed by the absence of detectable acylenzyme accumulation with all the tested substrates, with the sole exception of cefoxitin.

scholarly journals The active site of the P99 β-lactamase from Enterobacter cloacae

1984 ◽  
Vol 223 (1) ◽  
pp. 271-274 ◽  
Author(s):  
B Joris ◽  
J Dusart ◽  
J M Frere ◽  
J van Beeumen ◽  
E L Emanuel ◽  
...  
Keyword(s):  
Active Site ◽  
Enterobacter Cloacae ◽  
Beta Lactamase ◽  
Serine Residue ◽  
Class C

Labelling the beta-lactamase of Enterobacter cloacae P99 with a poor substrate or a mechanism-based inactivator points to an active-site serine residue in a sequence closely resembling that of the ampC beta-lactamase. These results establish the P99 enzyme as a class-C beta-lactamase, and the concurrence of the two approaches helps to confirm the reliability of determining active-site sequences with the aid of mechanism-based inactivators.

scholarly journals The effect of replacing the conserved active-site residues His-264, Asp-312 and Arg-314 on the binding and catalytic properties of Escherichia coli citrate synthase

1994 ◽  
Vol 300 (3) ◽  
pp. 765-770 ◽  
Author(s):  
W J Man ◽  
Y Li ◽  
C D O'Connor ◽  
D C Wilton
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Citrate Synthase ◽  
Salt Bridge ◽  
Site Directed Mutagenesis ◽  
Active Site Residues ◽  
E Coli ◽  
Rate Limiting Step ◽  
Rate Limiting

The first step in the overall catalytic mechanism of citrate synthase is the binding and polarization of oxaloacetate. Active-site residues Arg-314, Asp-312 and His-264 in Escherichia coli citrate synthase, which are involved in oxaloacetate binding, were converted by site-directed mutagenesis to Gln-314, Asn-312 and Asn-264 respectively. The R314Q and D312N mutants expressed negligible overall catalytic activity at pH 8.0, the normal assay pH, but substantial activities for the partial reactions that reflect the cleavage and hydrolysis of the substrate intermediate citryl-CoA. However, when the pH was lowered to 7.0, the overall reaction of the mutants became significant, in contrast to the wild-type enzyme, whereas the two mutants exhibited reduced activities for the partial reactions. This result is consistent with the existence of a rate-limiting step between the two partial reactions for these mutants that is pH-dependent. The Km for oxaloacetate for the two mutants was increased 10-fold and was paralleled by an increase in the Km for citryl-CoA, whereas the Km for acetyl-CoA was increased only 2-fold. Overall, there was a striking parallel between the results obtained for these two mutants, which suggests that they are functionally linked in the E. coli enzyme. The equivalent of these two residues form a salt bridge in the pig heart citrate synthase crystal structure. The H264N mutant, in which the amide nitrogen of asparagine should mimic the delta-nitrogen of histidine, showed negligible activity in terms of both overall and partial catalysis, which may result from a hindrance of conformational change upon oxaloacetate binding. The affinity of this mutant for oxaloacetate appeared to be greatly reduced when investigated using indirect fluorescence and chemical modification techniques.

scholarly journals The β-lactamase of Enterobacter cloacae P99. Chemical properties, N-terminal sequence and interaction with 6β-halogenopenicillanates

1985 ◽  
Vol 228 (1) ◽  
pp. 241-248 ◽  
Author(s):  
B Joris ◽  
F De Meester ◽  
M Galleni ◽  
G Reckinger ◽  
J Coyette ◽  
...  
Keyword(s):  
Active Site ◽  
Chemical Properties ◽  
Enterobacter Cloacae ◽  
Order Rate Constant ◽  
Beta Lactamase ◽  
Serine Residue ◽  
Terminal Sequence ◽  
Beta Lactamases ◽  
A Value ◽  
K 12

The beta-lactamase of Enterobacter cloacae P99 consists of one polypeptide chain of Mr 39000 devoid of disulphide bridges and free thiol groups. It contains an unusually high proportion of tyrosine and tryptophan. The N-terminal sequence exhibits overlaps with the tryptic peptide obtained after labelling the active site with 6 beta-iodopenicillanate. The active-site serine residue is at position 64. The homology with the chromosomal beta-lactamase of Escherichia coli K 12 (ampC gene) is lower within the 25 residues of the N-terminal portion than around the active-site serine residue. The P99 beta-lactamase is inactivated by 6 beta-bromo- and 6 beta-iodo-penicillanate, with a second-order rate constant of 110-140M-1 X s-1 at 30 degrees C and pH 7.0, a value that is much lower than that observed with class-A beta-lactamases.

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.

scholarly journals Threonine 57 is required for the post-translational activation ofEscherichia coliaspartate α-decarboxylase

2014 ◽  
Vol 70 (4) ◽  
pp. 1166-1172 ◽  
Author(s):  
Michael E. Webb ◽  
Briony A. Yorke ◽  
Tom Kershaw ◽  
Sarah Lovelock ◽  
Carina M. C. Lobley ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Intramolecular Rearrangement ◽  
Directed Mutagenesis ◽  
Vitamin B ◽  
Biosynthesis Pathway ◽  
Serine Residue ◽  
Translational Activation

Aspartate α-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of β-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formedviathe intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads to changes in the degree of post-translational activation. The crystal structure of the site-directed mutant T57V is consistent with a non-rearranged backbone, supporting the hypothesis that Thr57 is required for the formation of the ester intermediate in activation.

scholarly journals Beyond the Active Site: Mechanistic Investigations of the Role of the Secondary Coordination Sphere and Beyond on Multi-Electron Electrocatalytic Reactions and Their Relations Between Kinetics and Thermodynamics

2020 ◽  
Author(s):  
Vincent Wang
Keyword(s):  
Thermodynamic Parameters ◽  
Coordination Sphere ◽  
Active Site ◽  
Rate Constants ◽  
Reduction Reaction ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Secondary Coordination Sphere ◽  
Catalytic Rate ◽  
Rate Limiting

<p>The development of an electrocatalyst with a rapid turnover frequency, low overpotential and long-term stability is highly desired for fuel-forming reactions, such as water splitting and CO<sub>2</sub> reduction. The findings of the scaling relationships between the catalytic rate and thermodynamic parameters over a wide range of electrocatalysts in homogeneous and heterogeneous systems provide useful guidelines and predictions for designing better catalysts for those redox reactions. However, such relationships also suggest that a catalyst with a high catalytic rate is typically associated with a high overpotential for a given reaction. Inspired by enzymes, the introduction of additional interactions through the secondary coordination sphere beyond the active site, such as hydrogen-bonding or electrostatic interactions, have been shown to offer a promising avenue to disrupt these unfavorable relationships. Herein, we further investigate the influence of these cooperative interactions on the faster chemical steps, in addition to the rate-limiting step widely examined before, for molecular electrocatalysts with the structural and electronic modifications designed to facilitate the dioxygen reduction reaction, CO<sub>2</sub> reduction reaction and hydrogen evolving reaction. Based on the electrocatalytic kinetic analysis, the rate constants for faster chemical steps and their correlation with the corresponding thermodynamic parameters are evaluated. The results suggest that the effects of the secondary coordination sphere and beyond on these fuel-forming reactions are not necessarily beneficial for promoting all chemical steps and no apparent relation between rate constants and thermodynamic parameters are found in some cases studied here, which may implicate the design of electrocatalysts in the future. Finally, these analyses demonstrate that the characteristic features for voltammograms and foot-of-the-wave-analysis plots are associated with the specific kinetic phenomenon among these multi-electron electrocatalytic reactions, which provides a useful framework to probe the insights of chemical and electronic modifications on the catalytic steps quantitatively (i.e. kinetic rate constants) and to optimize some of critical steps beyond the rate-limiting step.</p>

In vitro Mutagenesis of Human Dihydropteridine Reductase at the Active Site and at Altered Sites Found in the Reductases of Deficient Children

Pteridines ◽  
1996 ◽  
Vol 7 (4) ◽  
pp. 123-136 ◽  
Author(s):  
Hong-Ping Zhang ◽  
Nan Yang ◽  
Wilfred L. F. Armarego
Keyword(s):  
Active Site ◽  
Enzyme Activities ◽  
General Procedure ◽  
Maximum Velocity ◽  
Order Rate Constant ◽  
Site Directed Mutagenesis ◽  
In Vitro Mutagenesis ◽  
Dihydropteridine Reductase ◽  
Proton Source

Summary A general procedure for in vitro site-directed mutagenesis of the wild-type dihydropteridine reductase gene has been used successfully to make eight mutant proteins. Five mutations were at the active site, viz Tyrl50His, Tyrl50Ser, Tyrl50Phe, Tyr150Glu and Tyrl50Lys. The proteins were expressed as glutathione S-transferase fusion proteins from which the unconjugated reductases were obtained by thrombin cleavage. The kinetic parameters of the conjugated and unconjugated reductases were measured using natural quinonoid R-7,8(6H)-dihydrobiopterin and non-natural quinonoid RS-6-methyl-7,8(6H)-dihydropterin and NADH. The kcat (maximum velocity at saturating concentrations of substrates) and kcatl Km (first order rate constant at low concentration of substrates) values show that the phenolic OH of Tyr 150 was the most likely proton source to complete the hydride reduction of the quinonoid pterin cofactor. However in the absence of a proton source at residue 150, measurable enzyme activities were observed indicating that a proton was relayed via a water molecule(s) from some neighbouring acidic amino acid residue. Three mutant dihydropteridine reductases, which were found in defective children, have been similarly attempted, viz GlylSlSer, Gly23Asp and a threonine insertion at position 123. The enzyme activities of the first two mutant reductases were consistent with the severity of the disease. The unconjugated reductase from the third mutation could not be obtained due to proteolysis but the fusion protein was enzymically active.

scholarly journals Structure-Function Studies of Ser-289 in the Class C β-Lactamase from Enterobacter cloacae P99

1999 ◽  
Vol 43 (3) ◽  
pp. 543-548 ◽  
Author(s):  
Sonia Trépanier ◽  
James R. Knox ◽  
Natalie Clairoux ◽  
François Sanschagrin ◽  
Roger C. Levesque ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Catalytic Efficiency ◽  
Enterobacter Cloacae ◽  
Acid Group ◽  
Site Directed Mutagenesis ◽  
Side Chain ◽  
Class C ◽  
Three Dimensional Models ◽  
Hydrolytic Mechanism

ABSTRACT Site-directed mutagenesis of Ser-289 of the class C β-lactamase from Enterobacter cloacae P99 was performed to investigate the role of this residue in β-lactam hydrolysis. This amino acid lies near the active site of the enzyme, where it can interact with the C-3 substituent of cephalosporins. Kinetic analysis of six mutant β-lactamases with five cephalosporins showed that Ser-289 can be substituted by amino acids with nonpolar or polar uncharged side chains without altering the catalytic efficiency of the enzyme. These data suggest that Ser-289 is not essential in the binding or hydrolytic mechanism of AmpC β-lactamase. However, replacement by Lys or Arg decreased by two- to threefold the k cat of four of the five β-lactams tested, particularly cefoperazone, cephaloridine, and cephalothin. Three-dimensional models of the mutant β-lactamases revealed that the length and positive charge of the side chain of Lys and Arg could create an electrostatic linkage to the C-4 carboxylic acid group of the dihydrothiazine ring of the acyl intermediate which could slow the deacylation step or hinder release of the product.

scholarly journals The K1 β-lactamase of Klebsiella pneumoniae

1987 ◽  
Vol 243 (2) ◽  
pp. 561-567 ◽  
Author(s):  
B Joris ◽  
F De Meester ◽  
M Galleni ◽  
J M Frère ◽  
J Van Beeumen
Keyword(s):  
Klebsiella Pneumoniae ◽  
Active Site ◽  
Cysteine Residue ◽  
Klebsiella Aerogenes ◽  
Beta Lactamase ◽  
Serine Residue ◽  
Class A

beta-Lactamase K1 was purified from Klebsiella pneumoniae SC10436. It is very similar to the enzyme produced by Klebsiella aerogenes 1082E and described by Emanuel, Gagnon & Waley [Biochem. J. (1986) 234, 343-347]. An active-site peptide was isolated after labelling of the enzyme with tritiated beta-iodopenicillanate. A cysteine residue was found just before the active-site serine residue. This result could explain the properties of the enzyme after modification by thiol-blocking reagents. The sequence of the active-site peptide clearly established the enzyme as a class A beta-lactamase.

Site-directed mutagenesis of the cysteinyl residues and the active-site serine residue of bacterial D-amino acid transaminase

Biochemistry ◽  
1989 ◽  
Vol 28 (2) ◽  
pp. 505-509 ◽  
Author(s):  
M. Merola ◽  
A. Martinez del Pozo ◽  
H. Ueno ◽  
P. Recsei ◽  
A. Di Donato ◽  
...  
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Serine Residue
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