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Title: Investigation of the mechanism of action of papain
Author: Yuthavong, Y.
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 1969
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The mechanism of papain-catalysed hydrolyses of K-acyl-amino acid derivatives is known to involve initial formation of an enzyme-substrate complex, followed by acylation of the active-site thiol of Cys-25 by the substrate to give a covalent acyl-enzyme intermediate, and subsequent deacylation of this intermediate. This thesis is concerned with an investigation of the various steps in catalysis, mainly by a study of steady-state kinetics of hydrolyses of specific substrates. The acylation step for a number of anilide substrates was found to be completely or partially rate-determining. The acylation rate constants (k+2) and the substrate constants (Ks) can be evaluated from the determination of Michaelis-Menten parameters (k0 and Km). For hippuryl p-substituted anilides and closely related analogues, k0=k+2 and Km=Ks. For m- and p-substituted anilides of acetyl-L-phenylalanylglycine (APG), k+2 were found to be comparable with the deacylation rate (kd); k+2 and Ks were calculable once kd was known. The deacylation step for the ester substrates studied was found to be the rate-determining step (k0=kd). The specificity of the enzyme for the N-acyl moiety of the substrate is probably determined to a large extent by hydrophobic interactions between the enzyme and the substrate, since (k0/Km) for N-acylglycine esters and anilides increase in magnitude in the order acetyl- < benzoyl- < benzyloxycarbonyl < acetyl-L-phenylalanyl-glycine derivatives. The greater reactivity of APG esters and anilides, despite the fact that hydrophobicity of the acyl moiety is comparable with benzoyl- and benzyloxycarbonyl-glycyl moieties, however, indicates that polar interactions are also significant. This conclusion is supported by the observation of differences of values of analogues of hippuric acid esters and anilides, in which the amide link has been modified. These differences are not solely determined by the inherent electronic properties of the acyl group, as measured by hydroxide-ion-catalysed rates of p-nitrophenyl esters. The amide link of N-acylamino acid derivatives may interact through its carbonyl oxygen with a polar group of the enzyme, possibly the γ-amide group of Gln-19, and the interaction may be important for the catalytic activity. As has been found by other workers, the enzyme interacts specifically with the side chain of L-arginine and it has been found that (k0/Km) for α-N-benzoyl-DL-arginine p-nitroanilide is 150 times that for hippuryl p-nitroanilide. The values of Ks for hippuryl anilides are in the range of 2-60 mM; those for APG anilides, 1-4 mM. Binding specificity is not a reliable indicator of papain activity for a given substrate, although frequently Ks is smaller for a more specific substrate. Free energy of binding interactions must be utilised in order to bring the enzyme-substrate complex to an active state for subsequent reactions. Comparison of activity among inhibitors and substrates derived from APG, methoxycarbonyl-L-phenylalanyl-glycine and hippuric acid indicates that the enzyme-substrate complex is subject to a strain around the susceptible bond, the amount of which accounts for its lability. An 'induced-fit' mechanism, involving conformational adjustment of the enzyme in order to bring the catalytic groups into proper positions, does not provide a satisfactory explanation for the experimental results. Among individual rate steps represented by Ks, k+2 and kd, the best measure of specificity is k+2, as demonstrated by comparison between anilides of APG, hippuric acid and analogues. k+2 values for APG anilides are 2-20 sec-1, while for hippuryl anilides, 3-50 msec-1. The 1,000-fold difference is due to both enthalpy and entropy of activation, as concluded from studies of the effect of temperature on the acylation rates of APG p-chloroanilide, hippuryl p-chloroanilide and hippuryl p-bromoanilide. In contrast with k+2, kd is not very sensitive to the nature of the N-acyl moiety. For acylglycine esters kd values lie normally between 1 and 10 sec-1, although (k0/Km) may differ by a factor of more than 104. Therefore, like Ks, kd is not a good indicator of specificity. A Hammett plot of log k+2 versus andsigma; for hippuryl anilides gives a andrho; value of -1.2, indicating electrophilic catalysis in the acylation step. The same andrho; value is obtained from a similar Hammett plot for APG anilides. Electronic effects on k+2 for both series of substrates are therefore similar. It is proposed that in the acylation of papain by anilide substrates the leaving groups are protonated through general acid catalysis. The donated proton is probably that originally forming a hydrogen bond between the essential imidazole group of His-159 and the essential thiol of Cys-25, which therefore may attack the carbonyl carbon of the substrate in the form of a thiolate anion. Available results do not allow a determination of the exact sequence of these processes. From the principle of microscopic reversibility the same processes in reverse must take place in the deacylation step. Experiments on kinetics of papain-catalysed hydrolyses of methyl and p-nitrophenyl esters in the presence of nucleophiles (methanol, aminoacetonitrile, glycinamide) support the suggestion of various workers that the rate- determining step in deacylation is the general-base-catalysed nucleophilic attack of water or other nucleophiles on the thiolester intermediate. A tetrahedral intermediate formed is rapidly decomposed to give the acid or other acyl derivatives. For hippuryl-papain and possibly for other acyl-papains, experimental results on the effects of aminoacetonitrile can be interpreted as indicating the existence of an alternative pathway. This may involve an acyl transfer to another group of the enzyme, or a conformational change, followed by relatively rapid hydrolysis and aminolysis. The acylation step depends on two prototropic groups with pKa(app) of approximately 4.2 and 8.3. The pKa( values of these groups in the free enzyme, as determined by the pH profile of (k0/Km) for p-nitrophenyl esters and APG p-nitroanilide, are very similar to those in the enzyme-substrate complex, as determined by the pH profile of k+2 for APG p-nitroanilide. If the two pKa( values represent protonation and deprotonation of the hydrogen-bonded thiol-imidazole system respectively, the binding of the substrate cannot result in the destruction of this hydrogen bond. As expected, pK2 was not observed in deacylation of APG-papin. In the deacylation step, pK1 is sensitive to the nature of the acyl moiety and varies in the range 3.1-4.7. The pK1 value tends to be low for a specific substrate or a substrate with hydrophobic groups in the acyl moiety. This observation can be interpreted in terms of shielding of the thiol-imidazole system by the acyl moiety from hydronium ions. Apart from steric effects, shielding by hydrophobic groups also gives rise to a decrease in local dielectric constant, which could further depress the pKa of a cationic acid such as the thiol-imidazolium system.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available