scholarly journals Peptidyldiazomethanes. A novel mechanism of interaction with prolyl endopeptidase

1992 ◽  
Vol 283 (3) ◽  
pp. 871-876 ◽  
Author(s):  
S R Stone ◽  
D Rennex ◽  
P Wikstrom ◽  
E Shaw ◽  
J Hofsteenge
Keyword(s):  
Active Site ◽  
Kinetic Mechanism ◽  
Prolyl Endopeptidase ◽  
Binding Mechanism ◽  
Inhibitory Mechanism ◽  
Serine Residue ◽  
Mechanism Of Inhibition ◽  
Progress Curve ◽  
Mechanism Of Interaction ◽  
Kinetics Of

Peptidyldiazomethanes with proline in the P1 position were found to be competitive slow-binding inhibitors of prolyl endopeptidase. Progress-curve experiments monitoring the increase in the degree of inhibition with time indicated that the kinetic mechanism involved an initial complex that isomerized to form a tighter complex. Reversibility of the inhibited complex was demonstrated by monitoring the regain of enzyme activity after removal of free inhibitor and dilution into an assay containing competing substrate. The kinetics of the reversal of inhibition indicated a more complicated inhibitory mechanism involving more than one pathway for reversal of the tight complex. A slow-binding mechanism of inhibition has not been previously observed with peptidyldiazomethanes. Incorporation of [3H]Ac-Ala-Ala-Pro-diazomethane into prolyl endopeptidase was observed after denaturation of the inhibited complex. The peptide labelled with [3H]Ac-Ala-Ala-Pro-diazomethane was isolated and found to contain the active-site serine residue.

scholarly journals Inactivation of prolyl endopeptidase by a peptidylchloromethane. Kinetics of inactivation and identification of sites of modification

1991 ◽  
Vol 276 (3) ◽  
pp. 837-840 ◽  
Author(s):  
S R Stone ◽  
D Rennex ◽  
P Wikstrom ◽  
E Shaw ◽  
J Hofsteenge
Keyword(s):  
Active Site ◽  
Kinetic Mechanism ◽  
Cysteine Residue ◽  
Prolyl Endopeptidase ◽  
Histidine Residue ◽  
Progress Curve ◽  
Kinetics Of ◽  
Active Site Histidine

The kinetics of inactivation of prolyl endopeptidase by acetyl-Ala-Ala-Pro-CH2Cl were studied by progress-curve methods in the presence of substrate. The kinetic mechanism was found to involve the formation of an initial complex between the enzyme and the chloromethane followed by an inactivation step. The substrate was shown to compete for the formation of the initial complex, indicating that binding at the active site was a prerequisite for inactivation. After reaction of the enzyme with [3H]acetyl-Ala-Ala-Pro-CH2Cl, it was possible to isolate five labelled peptides. Four of these peptides contained a cysteine residue as the site of modification, whereas the fifth peptide contained no cysteine and a histidine residue was identified as the site of modification. This residue (His-680) probably represents the active-site histidine of prolyl endopeptidase.

scholarly journals The kinetic mechanism of the major form of ox kidney aldehyde reductase with d-glucuronic acid

1982 ◽  
Vol 205 (2) ◽  
pp. 381-388 ◽  
Author(s):  
Ann K. Daly ◽  
Timothy J. Mantle
Keyword(s):  
Glucuronic Acid ◽  
Alternative Pathway ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Aldehyde Reductase ◽  
Major Form ◽  
Mechanism Of Inhibition ◽  
Steady State Kinetics ◽  
Inhibition Studies ◽  
Kinetics Of

The steady-state kinetics of the major form of ox kidney aldehyde reductase with d-glucuronic acid have been determined at pH7. Initial rate and product inhibition studies performed in both directions are consistent with a Di-Iso Ordered Bi Bi mechanism. The mechanism of inhibition by sodium valproate and benzoic acid is shown to involve flux through an alternative pathway.

scholarly journals Studying of the complexes product of the nerve agent Soman with the Butyrylcholinesterase and Acetylcholinesterase Enzymes

2008 ◽  
Vol 5 (2) ◽  
pp. 297-304
Author(s):  
Baghdad Science Journal
Keyword(s):  
Active Site ◽  
Unknown Function ◽  
Reaction Path ◽  
Nerve Agent ◽  
Serine Residue ◽  
Catalytic Function ◽  
Mechanism Of Inhibition ◽  
3D Geometry ◽  
Degradation Reaction ◽  
Hydrolysis Of

Cholinesterases are among the most efficient enzymes known. They are divided into two groups: acetylcholinesterase (AChE) involved in the hydrolysis of the neurotransimitter acetylcholine, and butyrylcholinesterase (BChE) of unknown function. Several crystal structures of the former have shown that the active site is located at the bottom of a deep and narrow gorge. Human BChE has attracted attention because it can hydrolyze toxic esters and nerve agents. Here we analyze the complexes of cholinesterase with soman by describing the 3D geometry of the complex, the active site, the changes happened through the inhibition and provide a description for the mechanism of inhibition. Soman undergoes degradation in the active site of the AChE and BChE. We calculate the energy of the products of the degradation reaction and suggest the reaction path. The product of the former reaction bind to serine residue in the active site and forming a stable bond and ends the catalytic function of the enzyme. This study has a useful role in the search of inhibitors to help in the treatment of Alzahimer's disease.

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 A comparative study of class-D β-lactamases

1993 ◽  
Vol 292 (2) ◽  
pp. 555-562 ◽  
Author(s):  
P Ledent ◽  
X Raquet ◽  
B Joris ◽  
J Van Beeumen ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Gene Sequence ◽  
Catalytic Properties ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Beta Lactamase ◽  
Serine Residue ◽  
Beta Lactamases ◽  
Class D ◽  
Burst Kinetics

Three class-D beta-lactamases (OXA2, OXA1 and PSE2) were produced and purified to protein homogeneity. 6 beta-Iodopenicillanate inactivated the OXA2 enzyme without detectable turnover. Labelling of the same beta-lactamase with 6 beta-iodo[3H]penicillanate allowed the identification of Ser-70 as the active-site serine residue. In agreement with previous reports, the apparent M(r) of the OXA2 enzyme as determined by molecular-sieve filtration, was significantly higher than that deduced from the gene sequence, but this was not due to an equilibrium between a monomer and a dimer. The heterogeneity of the OXA2 beta-lactamase on ion-exchange chromatography contrasted with the similarity of the catalytic properties of the various forms. A first overview of the enzymic properties of the three ‘oxacillinases’ is presented. With the OXA2 enzyme, ‘burst’ kinetics, implying branched pathways, seemed to prevail with many substrates.

scholarly journals Chemical modification of serine at the active site of penicillin acylase from Kluyvera citrophila

1991 ◽  
Vol 280 (3) ◽  
pp. 659-662 ◽  
Author(s):  
J Martín ◽  
A Slade ◽  
A Aitken ◽  
R Arche ◽  
R Virden
Keyword(s):  
Active Site ◽  
Tertiary Structure ◽  
Thiol Group ◽  
Iodoacetic Acid ◽  
Penicillin Acylase ◽  
Protein Product ◽  
Side Chain ◽  
Serine Residue ◽  
Conserved Sequence ◽  
Ester Substrate

The site of reaction of penicillin acylase from Kluyvera citrophila with the potent inhibitor phenylmethanesulphonyl fluoride was investigated by incubating the inactivated enzyme with thioacetic acid to convert the side chain of the putative active-site serine residue to that of cysteine. The protein product contained one thiol group, which was reactive towards 2,2′-dipyridyl disulphide and iodoacetic acid. Carboxymethylcysteine was identified as the N-terminal residue of the beta-subunit of the carboxy[3H]methylthiol-protein. No significant changes in tertiary structure were detected in the modified penicillin acylase using near-u.v. c.d. spectroscopy. However, the catalytic activity (kcat) with either an anilide or an ester substrate was decreased in the thiol-protein by a factor of more than 10(4). A comparison of sequences of apparently related acylases shows no other extensive regions of conserved sequence containing an invariant serine residue. The side chain of this residue is proposed as a candidate nucleophile in the formation of an acyl-enzyme during catalysis.

Oxidation of Olefins Representing Some Structural Units of GR-S

1952 ◽  
Vol 25 (1) ◽  
pp. 21-32 ◽  
Author(s):  
W. C. Warner ◽  
J. Reid Shelton
Keyword(s):  
Phenyl Group ◽  
Kinetic Mechanism ◽  
Oxidation Reactions ◽  
Chain Reaction ◽  
Instantaneous Rate ◽  
Initial Oxygen ◽  
Oxidation Of Olefins ◽  
A Minor ◽  
The Relationship ◽  
Kinetics Of

Abstract Three olefins were oxidized in the liquid phase with molecular oxygen to determine the kinetics of the oxidation reactions and the relationship to oxidation of rubber. The instantaneous rate of oxidation was found to be related to the analytically determined olefin and peroxide concentrations by the equation : Rate=k (unreacted olefin)(peroxide), where rate equals moles of oxygen per mole of original olefin per hour and the parentheses represent molarities. Presence of a phenyl group was found to affect k, but only in a minor way, indicating that the same fundamental kinetic mechanism applies in both aromatic and aliphatic olefins. The data are consistent with the general kinetic mechanism of Bolland involving oxygen attack at the alpha-methylenic group. However, it appears probable that initial oxygen attack can also occur at the double bond, resulting in the formation of a peroxide biradical, which may then react with other olefin molecules, initiating the usual chain reaction mechanism.

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