Chymotrypsin acylation

Hubbard and Kirsch (1972) have recently proposed that histidine may act as a nucleophile in a-chymotrypsin acylation reactions of esters having a good leaving group (jO-nitrophenol). This suggestion was based on a similarity in p-value for acylation by p-substituted nitrophenyl and dinitrophenyl benzoates and nucleophilic attack on these compounds by imidazole, in contrast with less positive p-values for hydroxide ion catalysis. Hammett p-values for hydrolysis of substituted phenyl esters are given in Table 6 and show little apparent trend. The values for hydroxide ion and alcoholate ions are... 

Fig. 28. Hydration dependence of chymotrypsin acylation. Dependence on relative humidity ( plp,) of the extent of conversion of the amide substrate, iV-succinyl-L-phenylalanine-p-nitroaniline (SPN), to the nitroaniline product and acyl enzyme, for a 1 1 SPN-o-chymotrypsin powder of nominal pH 7.5. Reaction time was 5-7 days. The weight percentages of sodium acetate present in the powder were curve 1, 0% curve 2, 6.4% curve 3, 12% curve 4, 17% and curve 5,56.5%. The ordinate, A = D416/DJ57, is a measure of the nitroaniline product. (From Khurgin etal., 1977.)...

Figure 4.34a. First stage in the hydrolysis of a peptide by chymotrypsin acylation. A tetrahedral transition state is formed, in which the peptide bond is cleaved. The amine component then rapidly diffuses away, leaving an acyl-enzyme intermediate, adapted from L. Stryer .

The rate of this reaction is 10 larger than for ethyl benzoate. The reaction is base-catalyzed and as expected in the presence of H20 instead of H2O solvent, the rate is reduced by a factor of at least two (/chAd = 3.5). Thus, proton-transfer occurs in the transition state and the analogy with a-chymotrypsin acylation is apparent. 

This derivative, prepared from an amino acid and the acyl azide, is selectively cleaved in 80% yield by chymotrypsin. ... 

Transition-state stabilization in chymotrypsin also involves the side chains of the substrate. The side chain of the departing amine product forms stronger interactions with the enzyme upon formation of the tetrahedral intermediate. When the tetrahedral intermediate breaks down (Figure 16.24d and e), steric repulsion between the product amine group and the carbonyl group of the acyl-enzyme intermediate leads to departure of the amine product. 

For many serine and cysteine peptidases catalysis first involves formation of a complex known as an acyl intermediate. An essential residue is required to stabilize this intermediate by helping to form the oxyanion hole. In cathepsin B a glutamine performs this role and sometimes a catalytic tetrad (Gin, Cys, His, Asn) is referred too. In chymotrypsin, a glycine is essential for stabilizing the oxyanion hole. 

Figure 7-7. Catalysis by chymotrypsin. The charge-relay system removes a proton from Ser 195, making it a stronger nucleophile. Activated Ser 195 attacks the peptide bond, forming a transient tetrahedral intermediate. Release of the amino terminal peptide is facilitated by donation of a proton to the newly formed amino group by His 57 of the charge-relay system, yielding an acyl-Ser 195 intermediate. His 57 and Asp 102 collaborate to activate a water molecule, which attacks the acyl-Ser 195, forming a second tetrahedral intermediate. The charge-relay system donates a proton to Ser 195, facilitating breakdown of tetrahedral intermediate to release the carboxyl terminal peptide .

However, diffusion of the reactive QM out of the enzyme active site is a major concern. For instance, a 2-acyloxy-5-nitrobenzylchloride does not modify any nucleophilic residue located within the enzyme active site but becomes attached to a tryptophan residue proximal to the active site of chymotrypsin or papain.23,24 The lack of inactivation could also be due to other factors the unmasked QM being poorly electrophilic, active site residues not being nucleophilic enough, or the covalent adduct being unstable. Cyclized acyloxybenzyl molecules of type a could well overcome the diffusion problem. They will retain both the electrophilic hydroxybenzyl species b, and then the tethered QM, in the active site throughout the lifetime of the acyl-enzyme (Scheme 11.1). This reasoning led us to synthesize functionalized... 

The 6-chloromethyl substituent (series 5 and 6) is required for the inactivation of a-chymotrypsin. Nevertheless, there is only a transient inactivation of HLE and thrombin through the formation of a stable acyl-enzyme in spite of the presence of this group as demonstrated by the spontaneous or hydroxylamine-accelerated reactivation of the treated enzymes (Scheme 11.3, pathway b).21 HLE is specifically inhibited when such an alkylating function is absent (series 7), always through the formation of a transient acyl-enzyme (Table 11.2). 

Since the imidazolide method proceeds almost quantitatively, it has been used for the synthesis of isotopically labeled esters (see also Section 3.2), and it is always useful for the esterification of sensitive carboxylic acids, alcohols, and phenols under mild conditions. This advantage has been utilized in biochemistry for the study of transacylating enzymes. A number of enzymatic transacylations (e.g., those catalyzed by oc-chymo-trypsin) have been shown to proceed in two steps an acyl group is first transferred from the substrate to the enzyme to form an acyl enzyme, which is then deacylated in a second step. In this context it has been shown[21] that oc-chymotrypsin is rapidly and quantitatively acylated by Af-fraw.s-cinnamoylimidazole to give /ra/w-cinnamoyl-a-chymotrypsin, which can be isolated in preparative quantities and retains its enzymatic activity (see also Chapter 6). 

Fig. 21 Rate-pH dependence of the acylation and deacylation steps in the chymotrypsin-catalysed hydrolysis of 4-nitrophenyl trimethylacetate...

The CD-mediated cleavage of p-N02C6H4NHC0CF3 proceeds by acyl transfer to a-CD. Since the trifluoracetyl-CD, so produced, hydrolyses fairly quickly even at pFI7, the overall reaction shows true catalysis. Thus, for the reaction in (27), a-CD behaves as a model enzyme and shows three of the features of chymotrypsin (i) it provides a hydrophobic binding site (ii) it catalyses the loss of leaving group and (iii) the reaction proceeds through an acyl intermediate (Komiyama and Bender, 1977 Bender and Komiyama, 1978). 

In the reaction with PNPA, myristoylhistidine [29] in a cationic micelle rapidly forms acetylimidazole as a fairly stable intermediate which is readily observable at 245 nm. On the other hand, a mixed micelle of [29] and N,N-dimethyl-N-2-hydroxyethylstearylammonium bromide [30] leads to the formation and decay of the intermediate, indicating that the acetyl group is transferred from imidazole to hydroxyl groups (Tagaki et al., 1977 Tagaki et al., 1979). This can be a model of cr-chymotrypsin which catalyses hydrolysis of PNPA (non-specific substrate) by initial acylation of the histidyl imidazole followed by acyl transfer to the seryl hydroxyl group (Kirsh and Hubbard, 1972), as indicated schematically in (12). 

Chymotrypsin catalysis takes place through a three-step process, equation (11), where ES is an enzyme substrate complex which breaks down to give an acylated enzyme intermediate, ES and Pj,... 

There is now convincing evidence that an acyl chymotrypsin intermediate is formed from both specific and non-specific substrates (Bender and Kezdy, 1964 Bender et al., 1964). This intermediate is undoubtedly an acylserine. Acyl- and phosphorylserine derivatives have been isolated and identified. In view of evidence such as a D2 O solvent isotope effect ( h2oAd2o) 2-3 for both acylation and deacylation (Bender and Hamilton, 1962), alcohol and amine nucleophiles showing little dependence on the p/iTa-value of the nucleophile in reaction with furoyl enzyme (Inward and Jencks, 1965), and the influence of increasing steric bulk in the acyl group (Fife and Milstien, 1967 Milstien and Fife, 1968,.1969), consistent... 

Nucleophilic attack on the acyl enzyme p-nitrophenoxycarbonyl-chymotrypsin, where the leaving group has a pATg-value comparable to that of histidine, takes place by a group in the active site which is most probably histidine (Fife et a/., 1972 Hutchins and Fife, 1972 Bender and Wedler, 1972), showing that nucleophilic attack is sterically possible [equation (14)]. p-Nitrophenolate ion is released... 

From the second-order rate constant for imidazole-catalysed cyclization of the ethyl ester (34) (8 x 10 M s" ) and the rate constant for acylation of a-chy mo trypsin by N-acetyltyrosine ethyl ester (1600 s ), it can be calculated that, in order to attain a rate constant of the magnitude seen in the a-chymotrypsin reaction, a neighbouring imidazole would have to possess an effective molarity of 200,000 M. An effective concentration of this magnitude is not unreasonable, but it is probable that other factors are also important in the enzymatic reaction. 

In accord with the above scheme, variation of the meta-substituent altered the rate of acylation but not that of deacylation. The reaction of equation (27) is similar to reaction of a-chymotrypsin with phenolic esters. 

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