Binding of the by-product analog benzylsuccinic acid by carboxypeptidase A

Byers, Larry D.; Wolfenden, Richard

doi:10.1021/bi00735a008

Cited by 216 publications

(126 citation statements)

References 47 publications

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“…D-Phenylalanine, a competitive inhibitor of ester hydrolysis in the present study, has several times been reported (15,16,21) to be also a competitive inhibitor for peptide hydrolysis. However, K , values for inhibition of peptidase activity are in the range 1 .O-2.0 mM, which is an order of magnitude larger than we find (0.15 mM) for inhibition of ester hydrolysis.…”

Section: Discussionsupporting

confidence: 66%

Reversible Inhibition of Carboxypeptidase A. IV. Inhibition of Specific Esterase Activity by Hippuric Acid and Related Species and other Amino Acid Derivatives and a Comparison with Substrate Inhibition

Bunting¹,

Myers²

1975

Can. J. Chem.

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. Can. J. Chem. 53,1993Chem. 53, (1975. The anions of each of the following carboxylic acids exhibit uncompetitive inhibition of the hydrolysis of 0-hippuryl-L-3-phenyllactic acid by bovine carboxypeptidase A at p H 7.5, 25", ionic strength 0.2: hippuric acid, p-chloro-and p-nitrohippuric acids, hippurylglycine, carbobenzoxyglycine, phenaceturic acid, N-(3-phenylpropanoyl)glycine, benzoxyacetic acid, 3-benzoylpropanoic acid, and 0-hippuryl-D-mandelic acid. In each case, this uncompetitive inhibition is consistent with the ordered binding of substrate and inhibitor to the enzyme; i.e. the inhibitor binds to E.S but not to the free enzyme. Evidence is presented for the binding site for uncompetitive inhibitors being the same as for inhibitory ester substrate molecules. Comparison of the specificities of uncompetitive inhibitors and esters which display substrate inhibition provides evidence for a critical conformational change which controls the binding of uncompetitive inhibitors and inhibitory substrate molecules.D-Phenylalanine, D-leucine, D-p-nitrophenylalanine, glycyl-L-tyrosine, glycyl-L-phenylalanine, and glycyl-L-leucine are competitive inhibitors of the enzymic hydrolysis of 0-hippuryl-L-3-phenyllactic acid, whereas the N-chloroacetyl derivatives of L-tyrosine, L-phenylalanine, and L-leucine are noncompetitive inhibitors. For the above D-amino acids, glycyl dipeptides, and N-chloroacetyl amino acids, the phenylalanine derivative in each case is a considerably stronger inhibitor than the corresponding leucine derivative. This preference is similar to that observed for the binding of peptide substrates but the reverse of that observed for ester substrates and simple mono-and dicarboxylate ion inhibitors.The peptide substrates carbobenzoxyglycylglycyl-L-phenylalanine and N-chloroacetyl-Lphenylalanine are noncompetitive inhibitors of the enzymic hydrolysis of 0-hippuryl-L-3-phenyllactic acid. This clearly demonstrates the presence of different ester and peptide binding sites in this enzyme, which is consistent with conclusions from recent studies in other laboratories. JOHN W. BUNTING et CHESTER D. MYERS. Can. J. Chem. 53,1993Chem. 53, (1975. A un p H de 7.5, une tempkrature de 25" et une force ionique de 0.2, les anions de chacun des acides carboxyliques suivants agissent comme inhibiteurs incompktitifs pour l'hydrolyse de I'acide 0-hippuryl L-phknyl-3 lactique par la carboxypeptidase A du boeuf: l'acide hippurique, les acides p-chloro-et p-nitrohippuriques, la hippurylglycine, la carbobenzoxyglycine, I'acide phknaceturique, la N-(phknyl-3 propanoyl) glycine, l'acide benzoxyacktique, I'acide benzoyl-3 propanoi'que et I'acide 0-hippuryl D-mandelique. Dans chaque cas, cette inhibition incompttitive est en accord avec une association ordonnke du substrat et de I'inhibiteur a l'enzyme; i.e. I'inhibiteur s'associe au E.S mais non a l'enzyme libre. On presente des resultats suggkrant que le site d'association des inhibiteurs incompktitifs est le mCme que le site pour les molkcules d'esters agissant comme substra...

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Section: Discussionsupporting

confidence: 66%

Reversible Inhibition of Carboxypeptidase A. IV. Inhibition of Specific Esterase Activity by Hippuric Acid and Related Species and other Amino Acid Derivatives and a Comparison with Substrate Inhibition

Bunting¹,

Myers²

1975

Can. J. Chem.

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“…As part of of dicarboxylic acids. During the course of this our continuing investigation of the influence of work, reports (2,3) by L-benzylsuccinic acid ( K , = 4.5 x lo-' M at p H 7.5, 25"). Our present st~idy suggests that dicarboxylic acid anions, in general, are somewhat more potent inhibitors than might have been expected from analogy with related monocarboxylic acids, and furthermore, that the inhibition constants of these species display informative structural dependences.…”

mentioning

confidence: 97%

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

Bunting¹,

Myers²

1974

Can. J. Chem.

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. Can. J. Chem. 52, 2053Chem. 52, (1974. Reversible inhibition of the hydrolysis of 0-hippuryl-L-3-phenyllactic acid by carboxypeptidase A has been studied for a series of decarboxylic acids at 25', p H 7.5, and ionic strength 0.2. All inhibitors studied displayed either strictly competitive or partially conipetitive inhibition kinetics. For the series C0,H(CH2),,C02H, strictly competitive inhibition was observed for II = 1, 3, 4, 8, 10, whereas partially competitive inhibition occurs for 11 = 2, 5, 6, 7. A series of 1 I 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 n-parameter by the equation -log K, = 2.2571 + I .75; arylnialonic acids are poorer inhibitors than expected on the basis of their n-parameters, in accord with a similar observation for nionocarboxylic acids. Phthalic acid is a strictly competitive inhibitor (K, = 1.7 niM), 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 filmaric acid gives only 15% inhibition a t 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.Iz 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. On a Ctudit ['inhibition reversible de I'hydrolyse de I'acide 0-hippuryl phenyl-3 L-lactique par la carboxypeptidase A pour ilne serie d'acides dicarboxyliques a 25", a pH 7.5 et a ilne force ionique de0.2. Tousles inhibiteurs etudies presentent une cinetiqued'inhibition qili est soit strictement competitive soit partiellement conipetitive. On a observe une inhibition strictement conipetitive pour les series C 0 2 H (CH2),,C02H ou 11 = I , 3, 4, 8, 10, pendant qu'on a observe une inhibition partiellenient competitive pour 11 = 2, 5, 6, 7. Une serie de I1 acides maloniques substitues par des groupes alcoyles et aryles sont tous des inhibiteurs strictenient conipCtitifs; les constantes d'inhibition pour Line serie de six acides alcoylmaloniques sont relikes avec le parametre n de Hansch par l'equation -log Ki = 2.25 n + 1.75; les acides arylnialoniques sont des inhibiteurs plus faibles que ce que I'on s'attendait en se basant sur leurs paranietres n ce qui est en accord avec ilne observation similaire pour les acides monocarboxyliqi~es. L'acide phtalique est un inhibiteur strictenient competitif (Ki = 1.7 mM), tandis que les acides isonieres isophtaliques et tCrCphtaliqi~es produisent une inhibition relativement hible niCme a 0.1 iL1. On a aussi etudie I'acide ho...

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“…Compounds 1 through 6 ( Figure 1) were previously synthesized. 15,16 Tris, tri-fluoro acetic acid (TFA), and ammonium acetate were obtained from Fluka Chemie GmbH (Taufkirchen, Germany). Gel filtration columns (NAP™-5) containing Sephadex ® G-25 medium were supplied by Amersham Biosciences (Freiburg, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…11 CPA has been used as a model target enzyme for developing strategies for the design of new inhibitors of other zinc proteases of medicinal interest, such as angiotensin-converting enzyme and matrix metalloproteases. [12][13][14] CPA was selected as a model enzyme for this pilot study, and low-molecular weight competitive inhibitors 15,16 for the enzyme were combined to obtain the CPA inhibitor mix. These inhibitors bind to the active site of CPA noncovalently.…”

mentioning

confidence: 99%

A Method for Screening Enzyme Inhibitors Using Size Exclusion Chromatography and ESI-LC-MS/MS

Mathur¹,

Park²,

Hartmann³

2005

SLAS Discovery

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A pilot study was performed for the development of a method to screen compound libraries using an electrospray mass spectrometer interfaced with liquid chromatography (LC). The mixture of compounds was obtained by combining low-molecular weight inhibitors of carboxypeptidase A (CPA), a representative zinc-containing proteolytic enzyme. After the incubation of the mixture with CPA, the enzyme-bound compounds were separated by size exclusion chromatography (SEC) from unbound compounds. The separation of compounds was affected by LC. Three compounds were identified, which represent the tight binding inhibitors of the library. These compounds were quantitated using an automatic switching valve to avoid the interference of buffer salts with the detection of analytes. The quantitated amounts of the compounds were found to be in good accordance with the K i values. (Journal of Biomolecular Screening 2005:30-35)

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Binding of the by-product analog benzylsuccinic acid by carboxypeptidase A

Cited by 216 publications

References 47 publications

Reversible Inhibition of Carboxypeptidase A. IV. Inhibition of Specific Esterase Activity by Hippuric Acid and Related Species and other Amino Acid Derivatives and a Comparison with Substrate Inhibition

Reversible Inhibition of Carboxypeptidase A. IV. Inhibition of Specific Esterase Activity by Hippuric Acid and Related Species and other Amino Acid Derivatives and a Comparison with Substrate Inhibition

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

A Method for Screening Enzyme Inhibitors Using Size Exclusion Chromatography and ESI-LC-MS/MS

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