Reversible Inhibition of the Esterase Activity of Carboxypeptidase A by Carboxylate Anions

1973 ◽  
Vol 51 (16) ◽  
pp. 2639-2649 ◽  
Author(s):  
John W. Bunting ◽  
Chester D. Myers
Keyword(s):  
Competitive Inhibition ◽  
Hydrophobic Interactions ◽  
Enzyme Inhibitor ◽  
Inhibition Kinetics ◽  
Aromatic Rings ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Reversible Inhibition ◽  
Mixed Inhibition ◽  
Carboxylate Anions

Reversible inhibition of the hydrolysis of O-(hippuryl)-L-3-phenyllactic acid by carboxypeptidase A has been studied for 26 carboxylate ion inhibitors at 25°, pH 7.5, and ionic strength 0.2 (NaCl). Competitive inhibition, partially competitive inhibition, and mixed inhibition kinetics were observed. Within homologous series, strictly competitive inhibition by the lower members gave way to partially competitive inhibition with higher members, while homologs having very large hydrocarbon moieties displayed mixed inhibition kinetics. Partially competitive inhibition in this system is not consistent with a scheme involving only 1:1 enzyme–inhibitor complexes; rather, higher order enzyme–inhibitor complexes will be necessary for a complete description of the inhibition mechanism.Inhibition constants (log Ki) for the aliphatic carboxylate ions which are strictly competitive inhibitors, are closely correlated linearly with Hansch's π-parameter for hydrophobicity. This quantitatively confirms the importance of hydrophobic interactions between carboxypeptidase A and inhibiting ions. Carboxylate ions containing aromatic rings are less effective inhibitors than expected on the basis of the π-parameters of their hydrocarbon moieties. The dependence of log Ki on π in this system is unusually strong for a binding phenomenon, and suggests that an inhibitor-dependent conformational change may also be involved.

Reversible Inhibition of Carboxypeptidase A. II. Inhibition of Esterase Activity by Dicarboxylic Acid Anions

1974 ◽  
Vol 52 (11) ◽  
pp. 2053-2063 ◽  
Author(s):  
John W. Bunting ◽  
Chester D. Myers
Keyword(s):  
Dicarboxylic Acid ◽  
Competitive Inhibition ◽  
Dicarboxylic Acids ◽  
Competitive Inhibitor ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Monocarboxylic Acids ◽  
Reversible Inhibition ◽  
Competitive Inhibitors ◽  
Hydrolysis Of

Reversible inhibition of the hydrolysis of O-hippuryl-L-3-phenyllactic acid by carboxypeptidase A has been studied for a series of decarboxylic acids at 25°, pH 7.5, and ionic strength 0.2. All inhibitors studied displayed either strictly competitive or partially competitive inhibition kinetics. For the series CO2H(CH2)nCO2H, strictly competitive inhibition was observed for n = 1, 3, 4, 8, 10, whereas partially competitive inhibition occurs for n = 2, 5, 6, 7. A series of 11 alkyl- and aryl-substituted malonic acids were all strictly competitive inhibitors; for a series of six alkylmalonic acids the inhibition constants are correlated with the Hansch π-parameter by the equation –log K1 = 2.257π + 1.75; arylmalonic acids are poorer inhibitors than expected on the basis of their π-parameters, in accord with a similar observation for monocarboxylic acids. Phthalic acid is a strictly competitive inhibitor (K1 = 1.7 mM), whereas the isomeric isophthalic and terephthalic acids cause relatively little inhibition even at 0.1 M; maleic acid is a partially competitive inhibitor, whereas the isomeric fumaric acid gives only 15% inhibition at 0.1 M. Homophthalic acid and 2,2-dimethyl- and 3,3-dimethylglutaric acids were also investigated.The characteristics of partially competitive inhibition displayed by all dicarboxylic acids and also monomethyl succinate and succinamic acid are consistent with a scheme which assumes the formation of an E.I2 complex. The observed specificity of dicarboxylic acid binding is used to postulate a schematic diagram for binding of these species to the enzyme, and an interpretation of this diagram is suggested on the basis of the crystallographically determined structure of the enzyme.

Reversible Inhibition of Carboxypeptidase A. III. Inhibition of Specific Esterase Activity by Substituted Benzoate and Related Anions

1975 ◽  
Vol 53 (13) ◽  
pp. 1984-1992 ◽  
Author(s):  
John W. Bunting ◽  
Chester D. Myers
Keyword(s):  
Ionic Strength ◽  
Esterase Activity ◽  
Competitive Inhibition ◽  
Chemical Properties ◽  
Enzyme Molecule ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Reversible Inhibition ◽  
Noncompetitive Inhibition ◽  
Hydrolysis Of

The reversible inhibition of the hydrolysis of O-hippuryl-L-3-phenyllactic acid by bovine carboxypeptidase A, has been studied for a series of para-substituted benzoate ions (p-XC6H4-CO2−) at pH 7.5, 25°, ionic strength 0.2. For X = H, F, CN, NH2, CH3 competitive inhibition occurs, whereas non-competitive inhibition occurs for X = CF3, NO2, Cl, Br, (CH3)2N, CH3O, (CH3)2CH, (CH3)3C. For X = C2H5 mixed inhibition is observed and this can be separated into individual competitive and noncompetitive components. Uncompetitive inhibition occurs with X = I. The distinction between competitive and noncompetitive inhibition appears to depend on the size of X rather than on its chemical properties. The p-tolylacetate and 3-(p-tolyl)propanoate ions display partially competitive inhibition consistent with the formation of E.I2 species. The inhibition by the 3-(p-iodophenyl)propanoate ion is complex and depends on the binding of at least two inhibitor ions per enzyme molecule.

scholarly journals Characterization of bromosulphophthalein binding to human glutathione S-transferase A1-1: thermodynamics and inhibition kinetics

2004 ◽  
Vol 382 (2) ◽  
pp. 703-709 ◽  
Author(s):  
Doris KOLOBE ◽  
Yasien SAYED ◽  
Heini W. DIRR
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Competitive Inhibition ◽  
Hydrophobic Interactions ◽  
Titration Calorimetry ◽  
Inhibition Kinetics ◽  
High Affinity ◽  
Anionic Ligand ◽  
Catalytic Function ◽  
High Affinity Site

In addition to their catalytic functions, GSTs (glutathione S-transferases) bind a wide variety of structurally diverse non-substrate ligands. This ligandin function is known to result in the inhibition of catalytic function. The interaction between hGSTA1-1 (human class Alpha GST with two type 1 subunits) and a non-substrate anionic ligand, BSP (bromosulphophthalein), was studied by isothermal titration calorimetry and inhibition kinetics. The binding isotherm is biphasic, best described by a set of two independent sites: a high-affinity site and a low-affinity site(s). The binding stoichiometries for these sites are 1 and 3 molecules of BSP respectively. BSP binds to the high-affinity site 80 times more tightly (Kd=0.12 μM) than it does to the low-affinity site(s) (Kd=9.1 μM). Binding at these sites is enthalpically and entropically favourable, with no linkage to protonation events. Temperature- and salt-dependent studies indicate the significance of hydrophobic interactions in the binding of BSP, and that the low-affinity site(s) displays low specificity towards the anion. Binding of BSP results in the release of ordered water molecules at these hydrophobic sites, which more than offsets unfavourable entropic changes during binding. BSP inhibition studies show that the binding of BSP to its high-affinity site does not inhibit hGSTA1-1. This site, located near Trp-20, may be related to the buffer-binding site observed in GSTP1-1. The low-affinity-binding site(s) for BSP is most probably located at or near the active site of hGSTA1-1. Binding to this site(s) results in non-competitive inhibition with respect to CDNB (1-chloro-2,4-dinitrobenzene) (KiBSP=16.8±1.9 μM). Given the properties of the H site and the relatively small size of the electrophilic substrate CDNB, it is plausible that the active site of the enzyme can simultaneously accommodate both BSP and CDNB. This would explain the non-competitive behaviour of certain inhibitors that bind the active site (e.g. BSP).

Inhibition kinetics for propionate degradation using propionate-enriched mixed cultures

1998 ◽  
Vol 38 (8-9) ◽  
pp. 443-451 ◽  
Author(s):  
S. H. Hyun ◽  
J. C. Young ◽  
I. S. Kim
Keyword(s):  
Phase I ◽  
Competitive Inhibition ◽  
Gas Production ◽  
Phase Iii ◽  
Utilization Rate ◽  
Inhibition Kinetics ◽  
Wide Range ◽  
Propionate Degradation ◽  
Acetate Utilization ◽  
Velocity Coefficient

To study propionate inhibition kinetics, seed cultures for the experiment were obtained from a propionate-enriched steady-state anaerobic Master Culture Reactor (MCR) operated under a semi-continuous mode for over six months. The MCR received a loading of 1.0 g propionate COD/l-day and was maintained at a temperature of 35±1°C. Tests using serum bottle reactors consisted of four phases. Phase I tests were conducted for measurement of anaerobic gas production as a screening step for a wide range of propionate concentrations. Phase II was a repeat of phase I but with more frequent sampling and detailed analysis of components in the liquid sample using gas chromatography. In phase III, different concentrations of acetate were added along with 1.0 g propionate COD/l to observe acetate inhibition of propionate degradation. Finally in phase IV, different concentrations of propionate were added along with 100 and 200 mg acetate/l to confirm the effect of mutual inhibition. Biokinetic and inhibition coefficients were obtained using models of Monod, Haldane, and Han and Levenspiel through the use of non-linear curve fitting technique. Results showed that the values of kp, maximum propionate utilization rate, and Ksp, half-velocity coefficient for propionate conversion, were 0.257 mg HPr/mg VSS-hr and 200 mg HPr/l, respectively. The values of kA, maximum acetate utilization rate, and KsA, half-velocity coefficient for acetate conversion, were 0.216 mg HAc/mg VSS-hr and 58 mg HAc/l, respectively. The results of phase III and IV tests indicated there was non-competitive inhibition when the acetate concentration in the reactor exceeded 200 mg/l.

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.

S-Adenosylethionine as an inhibitor of tRNA methylation

1969 ◽  
Vol 47 (5) ◽  
pp. 561-565 ◽  
Author(s):  
B. G. Moore ◽  
R. C. Smith
Keyword(s):  
Escherichia Coli ◽  
Preliminary Data ◽  
Kinetic Data ◽  
Competitive Inhibition ◽  
Inhibition Kinetics ◽  
Inhibition Reaction ◽  
Inhibition Pattern ◽  
Methylase Activity

S-Adenosylethionine was shown to inhibit the methylation of tRNA by S-adenosylmethionine. Lineweaver–Burk plots of kinetic data suggested that the inhibition was competitive. Another known inhibitor of tRNA methylation, adenine, also exhibited competitive inhibition kinetics. A derivative of S-adenosylethionine, ethylthioadenosine, also inhibited the reaction and displayed a competitive inhibition pattern. These data were obtained using Escherichia coli K12W6 as a source of methyl-deficient tRNA and methylase enzymes. Preliminary data indicate that S-adenosylethionine is even a better inhibitor of rat methylases.Methylase activity of ethionine-fed rats was elevated, which suggested that the inhibition reaction with S-adenosylethionine and the increased methylase activity may proceed by two different pathways.

Elastin-derived peptide binding to a hydrophobic domain on neutrophil elastase

1994 ◽  
Vol 72 (9-10) ◽  
pp. 419-427 ◽  
Author(s):  
Suresh C. Tyagi ◽  
Sanford R. Simon
Keyword(s):  
Molecular Weight ◽  
Neutrophil Elastase ◽  
Protein Fraction ◽  
Competitive Inhibition ◽  
Hydrophobic Interactions ◽  
Human Aorta ◽  
Low Molecular Weight ◽  
Human Neutrophil Elastase ◽  
Amidolytic Activity ◽  
Hydrophobic Domain

To understand the contributions of binding of elastin to domains removed from the active site of neutrophil elastase, we isolated an elastin-derived peptide (EDP) fraction, which we have previously shown was tightly linked to neutrophil elastase after prolonged digestion of elastin but which can be released from the enzyme with hydroxylamine. Elastin from human aorta was incubated with human neutrophil elastase under conditions favoring proteolysis. Low molecular weight species, including free EDP, were separated from the protein fraction by a small centrifuged gel filtration column. The high molecular weight protein fraction was subjected directly to 0.5 M hydroxylamine. The reaction mixture was then fractionated on a phosphocellulose column using an ionic gradient. A fraction was collected that exhibited fluorescence with a peak at ~410 nm when excited at 320 nm, indicating the presence of desmosine and (or) isodesmosine. A second peak with amidolytic activity towards methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroaniline (MeOSucAAPVpNa), but no fluorescence at 410 nm was also detected at the same elution volume where free elastase appeared. After removal of low molecular weight digestion products but prior to treatment with hydroxylamine, the putative elastase–EDP complex possessed no amidolytic activity towards MeOSucAAPVpNa. When the liberated EDP was added to elastase in an amidolytic assay, the EDP behaved as only a partial noncompetitive inhibitor [Formula: see text], but bound with high affinity to neutrophil elastase [Formula: see text], as detected by its ability to quench elastase endogenous fluorescence. The complete emission spectrum of the mixture of elastase and EDP obtained at excitation wavelengths specific for tryptophan and desmosine/isodesmosine suggests that the EDP was in a hydrophobic environment which was close to at least one of the three tryptophan residues in the enzyme. Based on fluorescence energy transfer, we have estimated a distance between the elastase and EDP of ~10 ± 3 Å (1 Å = 0.1 nm) during elastinolysis. This pattern of binding to a hydrophobic site on neutrophil elastase without competitive inhibition of amidolytic activity was consistent with the importance of hydrophobic interactions between neutrophil elastase and elastin within a region of the enzyme removed from the active site.Key words: proteinase, elastase, elastin, extracellular matrix, elastin-derived peptide.

A Preference for Edgewise Interactions between Aromatic Rings and Carboxylate Anions:  The Biological Relevance of Anion−Quadrupole Interactions

2007 ◽  
Vol 111 (28) ◽  
pp. 8242-8249 ◽  
Author(s):  
Michael R. Jackson ◽  
Robert Beahm ◽  
Suman Duvvuru ◽  
Chandrasegara Narasimhan ◽  
Jun Wu ◽  
...  
Keyword(s):  
Aromatic Rings ◽  
Quadrupole Interactions ◽  
Biological Relevance ◽  
Carboxylate Anions
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