The Primary Specificity of α-Chymotrypsin. Acylated Amino Acid Esters with Normal Alkyl Side Chains<sup>1</sup>

Jones, J. Bryan; Kunitake, Toyoki; Niemann, Carl; Hein, George E.

doi:10.1021/ja01086a029

Cited by 59 publications

(26 citation statements)

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“…Substrates with similar chemical structure but different hydrophobic substituents react at different rates with chymotrypsin as the catalyst. A study of the side-chain specificity for a chymotrypsin-catalyzed hydrolysis of a series of a-N-acetyl-L-amino acid methyl esters showed kinetic constants (keat/Km(app)) increasing from 0.42 for hydrogen to 8,200 for n-C5H1 and then decreasing to 2,100 for n-C6H13 (Jones et al, 1965). There is increased enzyme-substrate binding as the hydrophobicity of the substrate increases with increasing side-chain length up to five carbons, but the six-carbon side chain is less well accommodated because of steric restrictions at the hydrophobic site of the enzyme (Zeffren 8c Hall, 1973).…”

Section: Can Enzymes With Partial Substrate Specificity Explain Chemomentioning

confidence: 99%

Chemosyndromic Variation in Lichens

Culberson¹,

Culberson²

1976

Systematic Botany

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Section: Can Enzymes With Partial Substrate Specificity Explain Chemomentioning

confidence: 99%

Chemosyndromic Variation in Lichens

Culberson¹,

Culberson²

1976

Systematic Botany

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“…Second-order polynomials were fitted to the first 15-20 data points (corresponding to 10-20°/0 of the complete progression curve) and the initial rates vo determined from the derivatives at zero time. The parameters heat and Km of the Michaelis-Menten equation (4) and their standard deviations were determined by iterative regression on Eqn (4) using first estimates of the parameters determined by linear regression on the reciprocal form (vo ws wo/[A]) of Eqn (4).…”

Section: Kinetic Analysismentioning

confidence: 99%

Kinetic Investigation of the α‐Chymotrypsin‐Catalyzed Hydrolysis of Peptide Substrates

Bizzozero¹,

Baumann²,

Dutler³

1973

European Journal of Biochemistry

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A number of peptide substrates of the general structure Ac-Lxn-. . . -Lxz-Lxl-Gly-NH, have been synthesized and their a-chymotrypsin-catalyzed hydrolyses studied. The acylation rate constants, E23 (= kcat), and the dissociation constants of the enzyme-substrate complexes, K J~A (= Km), have been determined using a modified pH-stat and a numerical method for the acquisition and processing of the data. On the basis of these constants a quantitative relationship between the peptide structure N-terminal to the cleaved bond and reactivity has been determined. The results are shown to be consistent with the enzyme-substrate interaction scheme proposed in 1971 by Segal et al. (Biochemistry 10, 3728). A comparison of the k , , l K g~ values indicates that the influence of a single structural change on the overall reactivity i s virtually independent of the nature of the remainder of the substrate. In addition a comparison of the k,, and K E A values shows that, in general, changes in substrate structure are mainly reflected by changes in k23 rather than in KEA. A few exceptions have been found: K E A or K E A and k,, change when glycine is introduced at the 22 position, when this glycine is replaced by alanine or when alanine is introduced at the 23 position.The observation that a-chymotrypsin has a marked side-chain specificity [l], in that it preferentially catalyzes the hydrolysis of peptide bonds C-terminal to aromatic amino-acid residues, led to the conclusion that the interactions between a single specific amino-acid residue and the active site of the enzyme are of primary importance in determining which peptide bond is to be cleaved. These interactions, which occur within what can be termed the primary interaction range, have therefore been investigated kinetically in detail using derivatives of single amino acids, mainly N-acylated esters, as model substrates I n the case of protein [8] and peptide [9] substrates, it appears that additional residues on both sides of the specific amino acid also interact with the active site. The importance of such additional interactions, which occur in the secondary interaction range, may be investigated by extending the kinetic studies to suitable peptide substrates. I n order to obtain kinetic data with peptides which is as accurate as that for ester substrates, it is necessary that only one peptide bond is hydrolyzed. For this purpose substrates of the general structure have been synthesized, where only Lxl is a specific amino acid residue. Thus it is expected that only the peptide bond between LZ1 and L,1 (indicated by the arrow) is cleaved during the measurements. In designing these substrates, it is important to select end groups Ex and E, which do not give rise to disturbing interactions with the enzyme.The pH-stat titration offers a simple method of analysis for ester hydrolysis. However, this method is technically more difficult to apply to peptide hydrolysis [lo], since the formation of amine product Em.

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“…Imperfection of the fit can be partly accounted for by the fact that the series is not homologous. Jones et al J18 1 calculated a similar value for chymotrypsin and this relates to a hydrophobic type of binding of the acyl moiety.…”

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confidence: 91%

Kinetic Investigations of Liver Microsomal Esterases with Oxazepam Esters

Maksay

Tegyey

Ötvös

1978

Hoppe-Seyler´s Zeitschrift für physiologische Chemie

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Hepatic microsomal esterases of the mouse responsible for the bioactivation of inactive (prodrug) esters of the centrally acting oxazepam were studied. The enzymes are situated on the cytoplasmic side of the microsomes. The esterases are partly solubilized and partly inactivated by homogenization in aqueous glycerol and treatment with deoxycholate. There is good correlation between the rates of hydrolysis and steric constants of the acyl moiety. Substrate binding increases to an optimum with the number of carbon atoms in the acyl moiety and is of hydrophobic nature. An attempt has been made to classify the esterases on the basis of the effect of inhibitors and activators. Kinetische Untersuchungen an der mikrosomalen Esterase aus Leber mittels OxazepamesternZusammenfassung: Untersucht wurden die mikrosomalen Esterasen aus Mäuseleber, die für die Bioaktivierung der inaktiven Oxazepamester (Vorstufen der zentral wirksamen Substanz) verantwortlich sind. Die Enzyme sind an der cytoplasmatischen Seite der Mikrosomen lokalisiert. Durch Homogenisieren in wäßrigem Glycerin und Behandlung mit Desoxycholat werden die Esterasen z.T. gelöst und z.T. inaktiviert. Zwischen den Hydrolysegeschwindigkeiten und den sterischen Konstanten des Acylanteiles besteht eine gute Korrelation. Die Substratbindung steigt mit der Zahl der Kohlenstoffatome im Acylanteil bis zu einem Optimum und ist von hydrophober Natur. Es wurde versucht, die Esterasen nach ihrer Beeinflußbarkeit durch Inhibitoren und Aktivatoren einzuteilen.

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The Primary Specificity of α-Chymotrypsin. Acylated Amino Acid Esters with Normal Alkyl Side Chains¹

Cited by 59 publications

References 1 publication

Chemosyndromic Variation in Lichens

Chemosyndromic Variation in Lichens

Kinetic Investigation of the α‐Chymotrypsin‐Catalyzed Hydrolysis of Peptide Substrates

Kinetic Investigations of Liver Microsomal Esterases with Oxazepam Esters

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The Primary Specificity of α-Chymotrypsin. Acylated Amino Acid Esters with Normal Alkyl Side Chains1

Cited by 59 publications

References 1 publication

Chemosyndromic Variation in Lichens

Chemosyndromic Variation in Lichens

Kinetic Investigation of the α‐Chymotrypsin‐Catalyzed Hydrolysis of Peptide Substrates

Kinetic Investigations of Liver Microsomal Esterases with Oxazepam Esters

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The Primary Specificity of α-Chymotrypsin. Acylated Amino Acid Esters with Normal Alkyl Side Chains¹