Chymotrypsin-Catalyzed Hydrolysis of m-, p-, and o-Nitroanilides of N-Benzoyl-L-tyrosine<sup>*</sup>

Bundy, Hallie F.; Moore, Sister Cecelia Louise

doi:10.1021/bi00866a056

Cited by 25 publications

(16 citation statements)

References 13 publications

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“…These investigations have shown that, in the chymotrypsin hydrolysis of specific anilides substituted in the aniline ring, the catalytic rate is decreased with increase in the electron withdrawal (@ < 0). These results, however, were obtained in studies that cover the low o--value region (6 < 0.4).There are some indications [5,8] that in the high o--value region the substituent dependence is the reverse of that observed in the low o--value region.The specificity of chymotrypsin arises from a hydrophobic interaction between enzyme and substrate [9]. For this reason chymotrypsin-specific ani- lides have poor solubility in buffer solutions.…”

mentioning

confidence: 70%

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Structure‐Activity Relationship in the Urokinase Hydrolysis of α‐N‐Acetyl‐l‐lysine Anilides

Petkov¹,

Christova²,

Pojarlieff³

et al. 1975

European Journal of Biochemistry

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The absence of both nonproductive binding and substrate activation and also the good solubility of the substrates make the urokinase-catalysed hydrolysis of specific anilides a very suitable reaction for substrate structure -enzyme activity studies.Derivatives of a-N-acetyl-L-lysine anilide with high a--value substituents in the aniline ring were synthesized. Rate constants k,,,, and apparent Michaelis-Menten constants K , (app.) are presented. From the substituent dependence of k,,,, and from the fact that k,,,. is 13 to 37 times smaller than the deacylation rate constant it is concluded that the rate-limiting step proceeds prior to deacylation.The catalytic rate constant k,,,. obeys a linear free-energy relationship of the Hammett type with e = +0.72. Two different mechanisms implied by the results obtained from the model reaction (specific base and general acid-base catalysed hydrolysis of N-acetylglycine anilides under extreme conditions) are proposed in order to account for this positive and low @-value. In the first mechanism the breakdown of an enzyme tetrahedral intermediate is rate-limiting, while in the second one its formation controls the overall rate. The discrimination between the two mechanisms, however, could not be found.The investigation of the electronic effects of the substituents in the substrate molecule on its reactivity in enzyme-catalysed reactions provides very valuable information for the mechanism of enzyme action. The responses to changes in the charge density at the reaction site have been exploited succesfully in the study of the chymotrypsin-catalysed hydrolysis of nonspecific [l] and specific [2,3] 0-acylphenols and specific anilides [4 -71. These investigations have shown that, in the chymotrypsin hydrolysis of specific anilides substituted in the aniline ring, the catalytic rate is decreased with increase in the electron withdrawal (@ < 0). These results, however, were obtained in studies that cover the low o--value region (6 < 0.4).There are some indications [5,8] that in the high o--value region the substituent dependence is the reverse of that observed in the low o--value region.The specificity of chymotrypsin arises from a hydrophobic interaction between enzyme and substrate [9]. For this reason chymotrypsin-specific ani- lides have poor solubility in buffer solutions. In order to increase the substrate solubility, organic solvents were used up to 22 % (v/v). Organic solvents, however, perturb the enzymatic reaction [5,8]. Moreover, in model reactions the effects of organic solvents are opposite in the low and high 6-value regions [lo]. This makes the rate measurements of the chymotrypsin hydrolysis very difficult and the separation of k,,, and K , somewhat imprecise [ll]. On the other hand, chymotrypsin substrates with a hydrophobic anilide portion bind predominantly in a nonproductive mode and then the variation in the Michaelis-Menten parameters are due to the substituent effects on the hydrophobic nature of the aniline ring [ll]. These complications could be avoided by...

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mentioning

confidence: 70%

“…In order to increase the substrate solubility, organic solvents were used up to 22 % (v/v). Organic solvents, however, perturb the enzymatic reaction [5,8]. Moreover, in model reactions the effects of organic solvents are opposite in the low and high 6-value regions [lo].…”

mentioning

confidence: 99%

Structure‐Activity Relationship in the Urokinase Hydrolysis of α‐N‐Acetyl‐l‐lysine Anilides

Petkov¹,

Christova²,

Pojarlieff³

et al. 1975

European Journal of Biochemistry

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“…This explanation, however, is based on work that covered only a small range of substituents in the ring (p-CH30, p-CH3, m-CH30, p-Cl, and m-Cl). Bundy and Moore (5), have found that for hydrolysis of substituted N-benzoyltyrosine anilides by a-chymotrypsin, the p-nitroanilide has a kcat twice that of the m-nitroanilide. Furthermore, the Km for the p-nitro compound is about three times that for the m-nitro.…”

Section: P1mentioning

confidence: 99%

Influence of Leaving-Group Electronic Effect on α-Chymotrypsin: Catalytic Constants of Specific Substrates

Philipp

Pollack

Bender

1973

Proc. Natl. Acad. Sci. U.S.A.

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Rate constants and binding constants for the a-chymotrypsin-catalyzed hydrolysis of N-acetyltyrosine, tryptophan, and phenylalanine anilides are presented. Both kcat and Km are independent of electronic effects in the substrate over a range of 9.8 orders of magnitude (as measured by pK of the leaving group). Similarly, Km is independent of charge and orientation about the a-carbon for various substrates and pseudo-substrates. These results are not consistent with the pretransition state protonation hypothesis; instead, they are discussed in terms of a tetrahedral intermediate that is thermodynamically less stable than the Michaelis complex.Several workers have studied the substituent dependence of a-chymotrypsin-catalyzed hydrolysis of anilides, but the results are inconclusive. It has been shown (1, 2) that for N-acetyltyrosine anilides substituted in the aniline ring, electron-donating substituents enhance kcat, the rate constant for acylation (p = -2.0). Binding of the anilide to the enzyme, however, is inhibited by electron-withdrawing groups, although a plot of Km versus a gives only a fair correlation (p = about -2) Km kcat E + S ES -ES' -E + P2 P1In order to account for these results, it was proposed that protonation of the leaving aniline occurs in a rapid preequilibrium before the rate-determining step of the reaction (2-4). The pretransition state protonation theory accounts for the accelerating effect of electron-donating groups in the ring by the formation of a positive charge on the amide nitrogen adjacent to the ring. This explanation, however, is based on work that covered only a small range of substituents in the ring (p-CH30, p-CH3, m-CH30, p-Cl, and m-Cl). Bundy and Moore (5), have found that for hydrolysis of substituted N-benzoyltyrosine anilides by a-chymotrypsin, the p-nitroanilide has a kcat twice that of the m-nitroanilide. Furthermore, the Km for the p-nitro compound is about three times that for the m-nitro. These results are not in accord with the pretransition state protonation theory, since a p-nitro group is more electron-withdrawing than is a m-nitro substituent. In addition, Inagami, Patchornik and York,(3) found that, in the N-acetyltyrosine system, the p-nitroanilide showed a large positive deviation from the correlation for both kcat and Km.Several explanations may be advanced for the apparent deviation of the nitro-substituted compounds. The first is that 517 the nitro substituent may be anomalous, as suggested by Inagami (3). The second is that there may be a change in mechanism so that the leaving group in the case of the nitroanilides is not the neutral aniline molecule, but is rather the negatively-charged anilide ion. A model for this process may be seen in previous work from these laboratories in which it was found that the hydroxide ion-catalyzed hydrolysis of ringsubstituted acetanilides occurs through a pathway involving protonation of the amide nitrogen for all compounds except p-nitroacetanilide and p-formylacetanilide. For these two compounds, the leaving g...

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“…For example, although a good correlation exists between the acylation rates (keat) of chymotrypsin by a series of ring substituted N-acetyl-tyrosine anilides and o-, the rate constant for the corresponding p-nitroanilide is fasted than predicted by a factor of over 100 [3]. In addition, acylation of chymotrypsin by nitro-substituted N-benzoyltyrosine anilides shows a rate enhancement as electron-withdrawal increases [5], whereas for other substituents the rate is increased by electron-donating groups [2][3][4].…”

Section: Introductionmentioning

confidence: 98%

“…They have been used as substrates for trypsin [1 ], chymotrypsin [2][3][4][5], elastase [6,7], aminopeptidase M [8], papain [1,9] and subtilisn [10]. It is unclear, however, whether p-nitroanilides are normal substrates for these enzymes.…”

Section: Introductionmentioning

confidence: 99%

On the use of p‐nitroanilides as substrates for proteolytic enzymes

Pollack

Dumsha

1974

FEBS Letters

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Chymotrypsin-Catalyzed Hydrolysis of m-, p-, and o-Nitroanilides of N-Benzoyl-L-tyrosine^*

Cited by 25 publications

References 13 publications

Structure‐Activity Relationship in the Urokinase Hydrolysis of α‐N‐Acetyl‐l‐lysine Anilides

Structure‐Activity Relationship in the Urokinase Hydrolysis of α‐N‐Acetyl‐l‐lysine Anilides

Influence of Leaving-Group Electronic Effect on α-Chymotrypsin: Catalytic Constants of Specific Substrates

On the use of p‐nitroanilides as substrates for proteolytic enzymes

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Chymotrypsin-Catalyzed Hydrolysis of m-, p-, and o-Nitroanilides of N-Benzoyl-L-tyrosine*

Cited by 25 publications

References 13 publications

Structure‐Activity Relationship in the Urokinase Hydrolysis of α‐N‐Acetyl‐l‐lysine Anilides

Structure‐Activity Relationship in the Urokinase Hydrolysis of α‐N‐Acetyl‐l‐lysine Anilides

Influence of Leaving-Group Electronic Effect on α-Chymotrypsin: Catalytic Constants of Specific Substrates

On the use of p‐nitroanilides as substrates for proteolytic enzymes

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Chymotrypsin-Catalyzed Hydrolysis of m-, p-, and o-Nitroanilides of N-Benzoyl-L-tyrosine^*