that chymotrypsin (and trypsin), wh

that chymotrypsin (and trypsin), which were thus far considered as ‘proteinases’ specifically splitting large proteins, were actually able to cleave ‘internal’ peptide bonds of short peptides also. Bergmann & Fruton[16] also found that various ester and amide derivatives of the
chymotrypsin-specific amino acids were also hydrolyzed effectively by the enzyme. Chymotrypsin A, either theα or the γ form, was investigated most extensively, but some comparative activity and specificity studies were also carried out with other variants. It was agreed that
chymotrypsin Aπis the most active form, and that further autolysis results in a gradual decrease of the catalytic activity[12,14]. It was also noted that chymotrypsin B, in
sharp contrast with chymotrypsin A, splits acyl-tryptophan esters very slowly[20]. Indeed, our recent comparison of the substrate specificities of cattle chymotrypsin A and recombinant rat chymotrypsin B has revealed that the former is about 100-fold more active on the substrate
Suc-Ala-Ala-Pro- Trp-NHMec (see below) than rat chymotrypsin B (Huda´ky, P., Kaslik, G., Szila´gyi, L., Venekei, I., Gra´f, L. unpublished results). The hydrolysis of amide and ester substrates by chymotrypsin is a three-step process in which an enzyme
substrate complex and an acyl enzyme intermediate are formed [21] (Figure 582.2). The first evidence for this
mechanism was reported by Hartley & Kilby[22] who observed a rapid initial burst in the liberation ofp-nitrophenol when chymotrypsin was mixed with excess p-nitrophenyl acetate or p-nitrophenyl ethyl carbonate. They postulated that initially the ester rapidly acylated
the enzyme in a mole-to-mole ratio, and that the rate of subsequent substrate turnover was limited by the slow hydrolytic deacylation of the enzyme. The existence of the acyl enzyme intermediate was ultimately proven by the isolation and crystallization of several
stable forms such as indolylacryloyl-chymotrypsin [23], tosyl-chymotrypsin (2CHA; [24])and two photoreversible cinnamoyl-chymotrypsins[25]. This later work is especially interesting because, due to the special structure of the bound inhibitor, light-induced cis
trans isomerization
increases the rate of deacylation by several orders of magnitude. Photoirradiaton of the inhibited chymotrypsin crystals triggers deacylation, so that the process can be directly studied by X-ray crystallography [26]. Furthermore, the formation of acyl enzyme intermediates in the pathway of amide hydrolysis was also deduced by nucleophile partitioning experiments[27]. Recently, careful analysis of the X-ray structure of γ-chymotrypsin has Slow Cys 1 Asn 245 Cys
1 Chymotrypsinogen autolysis activation activation and autolysis Tyr Neochymotrypsinogen
Ala 146 149 Asn 245 Cys 1 Tyr α-Chymotrypsin Ala 146 149 Leu Ile 13 16 Asn 245 Fast autolysis autolysis Cys 1 Tyr γ-Chymotrypsin Ala 146 149 Leu Ile 13 16 Asn 245 Cys 1 δ-Chymotrypsin
π-Chymotrypsin Leu Ile 13 16 Asn 245 Cys 1 Arg Ile 15 16 Asn 245 FIGURE 582.1 ‘Slow’ and ‘fast’ activation of chymotrypsinogen. E-OH + RCOX E-OH.RCOX E-OH + RCOOH H2O k3 E-OC k2 XH
k+1
k–1
O
R
FIGURE 582.2 Kinetic scheme of chymotrypsin action. In a rapid equilibrium process (Ks5k11
/k
1) the enzyme forms a Michaelis complex with an amide or ester substrate which is cleaved in the next step (characterized by rate constantk2) to form an acyl-enzyme intermediate and an amide or alcohol. The rate constant of the hydrolysis of the acyl-enzyme isk3.
2627 Clan PA
S1 |582. Chymotrypsin revealed that this form is an acyl enzyme complex of
α-chymotrypsin with its autolysis product (Dixon & Matthews[28]: structure 1GCT; Dixon etal.[29]: structures 2GCT, 3GCT; Hareletal.[30]: structure 8GCH).In hexane,the tetrahedral intermediate of the reaction was also observed (Yennawaret al.[31]: 1GCM).
Besidestheir theoretical importance, burst substrates offer a simple and convenient way to determine the concentration of active enzyme by active site titration.
Initially, various p-nitrophenyl esters were used for this purpose, but more recently, substances with fluorogenic leaving groups, such as 4-methyl-umbelliferyl p-(N,N,N,-triethylammonium) cinnamate [32] havebeen developed, which provide a significantly higher sensitivity (20
50 pmol). A recently described active-site titration of chymotrypsin withα2-macroglobulin and HPLC may estimate proteinase activity with as little as 5
10 pmol
enzyme[33]. In the course of sequence analysis studies, chymotrypticdigests of many peptides and proteins have been examined in detail. In addition to hydrolysis at aromatic amino
acids and leucine, hydrolysis of bonds formed by asparagine, cysteic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine and valine have also been found.
Cleavage at these sites was not extensive, however, and depended strongly on the P1
0 residue ([34], and references therein). Chymotrypsin does not hydrolyze bonds formed
to the imino group of proline in proteins. Schellenberger et al. (1991) [35]performed quantitative structure/activity analysis of all available quantitative data on the chymotrypsin-catalyzed hydrolysis of amino acid and short peptide substrates. The substrates in the database span a range from the P5 to P3 0 positions. It was found that parameters for the P5, P4 and P3
0 subsites could be omitted from the calculations, which implied that interactions at these sites did not contribute significantly to the catalytic efficiency of chymotrypsin. The calculated P1 contributions to log (kcat/Km) gave linear correlation to the molar refraction of
the P1 side chain. (Molar refraction is the measure of the volume and polarizability of a substituent.) Substrate specificity mapping using all 400 possible dipeptides in membrane-bound form gave similar results[36]. The most common and convenient analytical technique for activity determination is spectrophotometry withp-nitroaniline as leaving group. Commercially available substrates include Suc-Phe-NHPhNO2 [37] and SucAla-Ala-Pro-Phe-NHPhNO2 [38]. Greater sensitivity can be achieved either by a fluorogenic leaving group (e.g. Suc-Ala-Ala-Pro-Phe-NHMec), with highly reactive thiobenzyl esters (Suc-Ala-Ala-Pro-Phe-SBzl; [39]) or with bioluminescence substrates like 6-(N-acetyl-L-phenylalanyl)-aminoluciferin[40]. Proline is commonly present at the P2 position in commerciallyavailable synthetic substrates (e.g. Suc-AlaAla-Pro-Phe-NHPhNO2). Depending on pH and temperature, about 85% of the Ala-Pro bond is trans, providing a good substrate for chymotrypsin, whereas thecisform is not a substrate. The activity of peptidylprolylcis
trans isomerase can therefore be measured in a coupled assay
with chymotrypsin[41,42]. Chymotrypsin is inhibited by general serine peptidase
inhibitors such as DFP [43], PMSF[44], DCI[45], leupeptin and chymostatin[46]. There are numerous naturally occurring protein inhibitors including turkey ovomucoid third domain[47], aprotinin [48], various serpins, marinostatin[49] and eglin c[50]. Novel synthetic
inhibitors include Z-Ala-Pro-Phe-glyoxal (Ki519 nm) and Z-Ala-Ala-Phe-glyoxal (Ki5344 nm) [51,52].The peptide IIe-Val-Asn-Gly-Glu-Glu-Ala-Val-Pro-Gly-SerTrp-Pro-Trp, corresponding to the N-terminal 14-mer of mature chymotrypsin A, is a highly potent, competitive
inhibitor [53]. Structural Chemistry Three amino acid residues, His57, Asp102 and Ser195,
the catalytic triad, are essential for peptide bond cleavage.
They are located at the entrance of a substrate-binding
pocket and their conservative arrangement is stabilized by
hydrogen bonds. These three amino acids are highly conserved in the sequences of the peptidases of family S1 (Chapter 559). A serine at position 214 is also highly conserved in the family, being present in all but three of almost 200 homologous bacterial and mammalian serine
proteinases [54,55,56]. Ser214 is hydrogen bonded to Asp102 and to the main-chain nitrogen atom of the scissile bond in the substrate. This residue contributes significantly to the polar environment that stabilizes the charge of the buried Asp102. A mutagenesis study performed on
trypsin supported the importance of the Ser214 function. This result and the invariance of this residue throughout the family led to the proposal that this residue might be considered as a fourth member of the catalytic triad[57]. The peptide amido groups of Gly193 and Ser195, in the
structural unit called the oxyanion hole, have important hydrogen bond donating interactions with the carbonyl group of the scissile peptide bond. These interactions are indispensable for catalysis since they orient the scissile bond NH to His57 and Ser195[58], initiating the formation of the tetrahedral intermediate. In the course of this process they dissipate the negative charge developing on the scissile bond carbonyl oxygen [15,59]. The interactions between the Sn,..S2, S1,S1 0 ,S2 0 ,..Sn 0 sites of the enzyme and the Pn,..P2, P1, P1
0 ,P20 ,..Pn 0 amino acid residues of the substrate ensure the precise alignment of the substrate to the catalytic triad and the oxyanion hole, and thereby the selectivity of catalysis.