scholarly journals Inactivation of penicillin acylase from Kluyvera citrophila by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline: a case of time-dependent non-covalent enzyme inhibition

1993 ◽  
Vol 291 (3) ◽  
pp. 907-914 ◽  
Author(s):  
J Martín ◽  
J M Mancheño ◽  
R Arche
Keyword(s):  
Enzyme Assay ◽  
Ph Dependence ◽  
Penicillin Acylase ◽  
Enzyme Inactivation ◽  
Time Dependent ◽  
Penicillin G ◽  
Reversible Binding ◽  
Ph Increase ◽  
Kinetics Of ◽  
Covalent Activation

Penicillin acylase (PA) from Kluyvera citrophila was inhibited by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a specific carboxy-group-reactive reagent. Enzyme activity progressively decreased to a residual value depending on EEDQ concentration. Neither enzymic nor non-enzymic decomposition of EEDQ is concomitant with PA inactivation. Moreover, enzyme re-activation is achieved by chromatographic removal of EEDQ, pH increase or displacement of the reagent with penicillin G. It was then concluded that PA inactivation is due to an equilibrium reaction. The kinetics of enzyme inactivation was analysed by fitting data to theoretical equations derived in accordance with this mechanism. Corrections for re-activation during the enzyme assay were a necessary introduction. The pH-dependence of the rate constant for EEDQ hydrolysis either alone or in the presence of enzyme was studied by u.v. spectroscopy. It turned out to be coincident with the pH-dependence of the forward and reverse rate constants for the inactivation process. It is suggested that previous protonation of the EEDQ molecule is required for these reactions to occur. The thermodynamic values associated with the overall reaction showed little change. Finally it is proposed that the inactivation of PA by EEDQ proceeds through a two-step reaction. The initial and rapid reversible binding is followed by a slow, time-dependent, non-covalent, reversible inactivating step. The expected behaviour in the case of enzyme modification by covalent activation of carboxy residues is also reviewed.

scholarly journals The kinetics of acylation and deacylation of penicillin acylase from Escherichia coli ATCC 11105: evidence for lowered pKa values of groups near the catalytic centre

1999 ◽  
Vol 338 (1) ◽  
pp. 235-239 ◽  
Author(s):  
Manuel MORILLAS ◽  
Martin L. GOBLE ◽  
Richard VIRDEN
Keyword(s):  
Rate Constant ◽  
Conformational Transition ◽  
Ph Dependence ◽  
Penicillin Acylase ◽  
Penicillin G ◽  
Pka Values ◽  
Energy Compensation ◽  
Guanidinium Group ◽  
Kinetics Of ◽  
Hydrolysis Of

Penicillin G acylase catalysed the hydrolysis of 4-nitrophenyl acetate with a kcat of 0.8 s-1 and a Km of 10 µM at pH 7.5 and 20 °C. Results from stopped-flow experiments fitted a dissociation constant of 0.16 mM for the Michaelis complex, formation of an acetyl enzyme with a rate constant of 32 s-1 and a subsequent deacylation step with a rate constant of 0.81 s-1. Non-linear Van't Hoff and Arrhenius plots for these parameters, measured at pH 7.5, may be partly explained by a conformational transition affecting catalytic groups, but a linear Arrhenius plot for the ratio of the rate constant for acylation relative to KS was consistent with energy-compensation between the binding of the substrate and catalysis of the formation of the transition state. At 20 °C, the pH-dependence of kcat was similar to that of kcat/Km, indicating that formation of the acyl-enzyme did not affect the pKa values (6.5 and 9.0) of an acidic and basic group in the active enzyme. The heats of ionization deduced from values of pKa for kcat, which measures the rate of deacylation, are consistent with α-amino and guanidinium groups whose pKa values are decreased in a non-polar environment. It is proposed that, for catalytic activity, the α-amino group of the catalytic SerB1 and the guanidinium group of ArgB263 are required in neutral and protonated states respectively.

scholarly journals Immobilization of alginate-PAC on Sepabeads EC-HA support

2011 ◽  
Vol 65 (4) ◽  
pp. 431-437 ◽  
Author(s):  
Milena Zuza ◽  
Nenad Milosavic ◽  
Zorica Knezevic-Jugovic
Keyword(s):  
Phenylacetic Acid ◽  
Immobilized Enzyme ◽  
Penicillin Acylase ◽  
Covalent Immobilization ◽  
Enzyme Inactivation ◽  
Penicillin G ◽  
Ph Profile ◽  
Covalently Linked ◽  
Hydrolysis Of ◽  
Derivatives Of

Penicillin acylase (PAC) is an important industrial enzyme for the production of many ?-lactam antibiotics. It is capable of catalyzing the hydrolysis of penicillin G (Pen G) to generate phenylacetic acid (PAA) and 6-aminopenicillanic acid (6-APA). In this paper, in order to prevent enzyme inactivation, an attempt of coupling enzyme modification and immobilization was presented. Chemical modification was promoted to introduce carbohydrate moiety into the PAC molecule, capable of being covalently linked to an amino support. This seems to provide a possibility to couple the enzyme without risking a reaction at the active site which might cause a loss of activity. PAC molecules were modified by cross-linking with polyaldehyde derivatives of alginate in order to add them new and useful functions. Immobilization of alginate-PAC on Sepabeads EC-HA was used as a model system in order to demonstrate the potential of this strategy. Optimal conditions for covalent immobilization of alginate-PAC from Escherichia coli on support Sepabeads EC-HA, were investigated. The immobilized enzyme was then characterized by evaluating the potential effects of immobilization on its thermal stability, temperature and pH profile in comparison with native non-modified PAC and modified non-immobilized PAC. The maximum amount of the alginate-PAC coupled on the dry support of 99 mg/g was satisfactory. Deactivation rate constants at 50 ?C for free PAC, alginate-PAC and alginate-PAC immobilized on Sepabeads EC-HA were 2,32; 50,65 and 1,68 h-1, respectively. Alginate-PAC and alginate-PAC immobilized on Sepabeads EC-HA had the same pH and temperature optimum as the native non-modified PAC.

scholarly journals β-lactamase inhibitors. The inhibition of serine β-lactamases by specific boronic acids

1988 ◽  
Vol 251 (2) ◽  
pp. 453-459 ◽  
Author(s):  
I E Crompton ◽  
B K Cuthbert ◽  
G Lowe ◽  
S G Waley
Keyword(s):  
Active Site ◽  
Ph Dependence ◽  
Boronic Acids ◽  
Penicillin G ◽  
Side Chain ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Kinetics Of ◽  
Kinetics Of Inhibition ◽  
Step Mechanism

Many beta-lactamases have active-site serine residues, and are competitively inhibited by boronic acids. Hitherto, the boronic acids used have lacked any structural resemblance to the substrates of beta-lactamases. Phenylacetamidomethaneboronic acid, trifluoroacetamidomethaneboronic acid and 2,6-dimethoxybenzamidomethaneboronic acid have now been synthesized. The first of these contains the side-chain moiety of penicillin G, and the last that of methicillin. The pH-dependence of binding of the first inhibitor to beta-lactamase I from Bacillus cereus revealed pK values of 4.7 and 8.2 for (presumably) active-site groups in the enzyme. The kinetics of inhibition were studied by cryoenzymology and by stopped-flow spectrophotometry. These techniques provided evidence for a two-step mechanism of binding of the first two boronic acids mentioned above to beta-lactamase I, and for benzeneboronic acid to a beta-lactamase from Pseudomonas aeruginosa. The slower step is probably associated with a change in enzyme conformation as well as the formation of an O-B bond between the active-site serine hydroxy group and the boronic acid.

scholarly journals Hydrogen exchange kinetics of human hemoglobins. The pH dependence of solvent accessibility in cyanomet-, oxy-, and deoxyhemoglobin.

1978 ◽  
Vol 253 (10) ◽  
pp. 3702-3707
Author(s):  
B.E. Hedlund ◽  
P.E. Hallaway ◽  
B.E. Hallaway ◽  
E.S. Benson ◽  
A. Rosenberg
Keyword(s):  
Hydrogen Exchange ◽  
Solvent Accessibility ◽  
Ph Dependence ◽  
Exchange Kinetics ◽  
Kinetics Of

scholarly journals In vivo analysis of the size- and time-dependent uptake of NaYF4:Yb,Er upconversion nanocrystals by pumpkin seedlings

2015 ◽  
Vol 3 (1) ◽  
pp. 144-150 ◽  
Author(s):  
J. Nordmann ◽  
S. Buczka ◽  
B. Voss ◽  
M. Haase ◽  
K. Mummenhoff
Keyword(s):  
Time Dependent ◽  
Upconversion Nanocrystals ◽  
In Vivo Analysis ◽  
Kinetics Of

We have investigated the kinetics of the uptake and the translocation of nanoparticles of different size in plants.

Dual kinetics of OATP2B1: Inhibitory potency and pH-dependence of OATP2B1 inhibitors

2021 ◽  
pp. 100416
Author(s):  
Ryo Sato ◽  
Takeshi Akiyoshi ◽  
Tokio Morita ◽  
Kazuhiro Katayama ◽  
Kodai Yajima ◽  
...  
Keyword(s):  
Ph Dependence ◽  
Inhibitory Potency ◽  
Kinetics Of

scholarly journals Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates.

1988 ◽  
Vol 8 (5) ◽  
pp. 1957-1969 ◽  
Author(s):  
R A Shapiro ◽  
D Herrick ◽  
R E Manrow ◽  
D Blinder ◽  
A Jacobson
Keyword(s):  
Steady State ◽  
Dictyostelium Discoideum ◽  
Mrna Decay ◽  
Decay Rates ◽  
Time Dependent ◽  
Translational Efficiency ◽  
Cdna Clones ◽  
Dependent Manner ◽  
Significant Difference ◽  
Kinetics Of

As an approach to understanding the structures and mechanisms which determine mRNA decay rates, we have cloned and begun to characterize cDNAs which encode mRNAs representative of the stability extremes in the poly(A)+ RNA population of Dictyostelium discoideum amoebae. The cDNA clones were identified in a screening procedure which was based on the occurrence of poly(A) shortening during mRNA aging. mRNA half-lives were determined by hybridization of poly(A)+ RNA, isolated from cells labeled in a 32PO4 pulse-chase, to dots of excess cloned DNA. Individual mRNAs decayed with unique first-order decay rates ranging from 0.9 to 9.6 h, indicating that the complex decay kinetics of total poly(A)+ RNA in D. discoideum amoebae reflect the sum of the decay rates of individual mRNAs. Using specific probes derived from these cDNA clones, we have compared the sizes, extents of ribosome loading, and poly(A) tail lengths of stable, moderately stable, and unstable mRNAs. We found (i) no correlation between mRNA size and decay rate; (ii) no significant difference in the number of ribosomes per unit length of stable versus unstable mRNAs, and (iii) a general inverse relationship between mRNA decay rates and poly(A) tail lengths. Collectively, these observations indicate that mRNA decay in D. discoideum amoebae cannot be explained in terms of random nucleolytic events. The possibility that specific 3'-structural determinants can confer mRNA instability is suggested by a comparison of the labeling and turnover kinetics of different actin mRNAs. A correlation was observed between the steady-state percentage of a given mRNA found in polysomes and its degree of instability; i.e., unstable mRNAs were more efficiently recruited into polysomes than stable mRNAs. Since stable mRNAs are, on average, "older" than unstable mRNAs, this correlation may reflect a translational role for mRNA modifications that change in a time-dependent manner. Our previous studies have demonstrated both a time-dependent shortening and a possible translational role for the 3' poly(A) tracts of mRNA. We suggest, therefore, that the observed differences in the translational efficiency of stable and unstable mRNAs may, in part, be attributable to differences in steady-state poly(A) tail lengths.

Oxypeucedanin is a mechanism-based inactivator of CYP2B6 and CYP2D6

2021 ◽  
Vol 22 ◽  
Author(s):  
Kehan Zhang ◽  
Yilin Li ◽  
Yao Fu ◽  
Tiantian Cui ◽  
Qian Wang ◽  
...  
Keyword(s):  
Pain Relief ◽  
Liver Microsomes ◽  
Reactive Intermediates ◽  
Enzyme Inactivation ◽  
Time Dependent ◽  
Reactive Metabolites ◽  
Angelica Dahurica ◽  
Dependent Inhibition ◽  
Microsomal Incubation

Background: Herbal medicine Angelica dahurica is widely employed for the treatment of rheumatism and pain relief in China. Oxypeucedanin is a major component of the herb. Objectives : The objectives of this study are aimed at the investigation of mechanism-based inactivation of CYP2B6 and CYP2D6 by oxypeucedanin, characterization of the reactive metabolites associated with the enzyme inactivation, and identification of the P450s participating in the bioactivation of oxypeucedanin. Methods : Oxypeucedanin was incubated with liver microsomes or recombinant CYPs2B6 and 2D6 under designed conditions, and the enzyme activities were measured by monitoring the generation of the corresponding products. The resulting reactive intermediates were trapped with GSH and analyzed by LC-MS/MS. Results : Microsomal incubation with oxypeucedanin induced a time-, concentration-, and NADPH-dependent inhibition of CYPs2B6 and 2D6 with kinetic values of KI/kinact 1.82 µM/0.07 min-1 (CYP2B6) and 8.47 µM/0.044 min-1 (CYP2D6), respectively. Ticlopidine and quinidine attenuated the observed time-dependent enzyme inhibitions. An epoxide and/or γ-ketoenal intermediate(s) derived from oxypeucedanin was/were trapped in microsomal incubations. CYP3A4 was the primary enzyme involved in the bioactivation of oxypeucedanin. Conclusion : Oxypeucedanin was a mechanism-based inactivator of CYP2B6 and CYP2D6. An epoxide and/or γ-ketoenal intermediate(s) may be responsible for the inactivation of the two enzymes.

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