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

Abstract

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...

Acylation is rate-limiting in glycosylasparaginase-catalyzed hydrolysis of N4-(4′-substituted phenyl)-l-asparagines

“…The charge (slope) on the nitrogen atom in the anilines containing electron-withdrawing groups is β lg = Ϫ0.25 (correlation coefficient r = 1.00 for the linear regression) and on the nitrogen atom in the anilines containing electrondonating groups is β lg = 0.43 (correlation coefficient r = 0.98 for the linear regression). These data are similar in magnitude to β lg ≈ 0.8 (calculated) 29 and β lg = 1.27 for electron-donating groups 31 in the reaction of chymotrypsin, and β lg ≈ Ϫ0.3 (calculated) 30 for electron-withdrawing groups in the reaction of urokinase.…”

“…27 Because the mechanism for GA has been proposed to be analogous to serine proteases, we compared our results with the most applicable results reported for rate-limiting acylation reactions utilizing anilides for chymotrypsin 28,29 and urokinase. 30 A biphasic Hammett plot for log (k cat ) vs. σ as in Fig. 2 was suggested for chymotrypsin by Bundy and Moore.…”

Nonlinear Free Energy Relationship in the General-Acid-Catalyzed Acylation of Rat Kidney γ-Glutamyl Transpeptidase by a Series of γ-Glutamyl Anilide Substrate Analogues

“…Free-energy relationships have also been used to characterize the transition state of the rate-limiting step of enzymatic reactions (), involving studies of substituent effects of parent carboxylic acid derivatives ( , ) or leaving groups ( − ). Some studies have used amino acid anilide substrates similar to those used herein to study the acylation reactions of cysteine proteases () and serine proteases ( , ). For example, the rate-limiting acylation of the serine protease chymotrypsin by amino acid anilides has been extensively studied and is of particular relevance to our current work.…”

“…For example, the rate-limiting acylation of the serine protease chymotrypsin by amino acid anilides has been extensively studied and is of particular relevance to our current work. Notably, it has been shown that the Hammett plot for this reaction curves upward ( − ), from a slope of ∼−2 with electron donating substituents (low σ) ( − ) to a slope of ∼0.7 for electron withdrawing substituents (high σ) ( , ). This curvature was explained in terms of the effect of the substituent on the degree of protonation of the aniline leaving group realized at the transition state ().…”

“…Some studies have used amino acid anilide substrates similar to those used herein to study the acylation reactions of cysteine proteases (41) and serine proteases (42,43). For example, the rate-limiting acylation of the serine protease chymotrypsin by amino acid anilides has been extensively studied and is of particular relevance to our current work.…”