Chymotrypsin active serine

Numbering corresponds to chymotrypsin with active serine at 195. 

The elucidation of the X-ray structure of chymotrypsin (Ref. 1) and in a later stage of subtilisin (Ref. 2) revealed an active site with three crucial groups (Fig. 7.1)-the active serine, a neighboring histidine, and a buried aspartic acid. These three residues are frequently called the catalytic triad, and are designated here as Aspc Hisc Serc (where c indicates a catalytic residue). The identification of the location of the active-site groups and intense biochemical studies led to several mechanistic proposals for the action of serine proteases (see, for example, Refs. 1 and 2). However, it appears that without some way of translating the structural information to reaction-potential surfaces it is hard to discriminate between different alternative mechanisms. Thus it is instructive to use the procedure introduced in previous chapters and to examine the feasibility of different... 

Sequences have been determined for plasminogen and bovine Factor XII, and they are not homologous with the other serine proteases. The amino-terminal sequence of Factor XII is homologous, however, with the active site of several naturally occurring protease inhibitors (11). Numbering corresponds to chymotrypsin with active serine at 195. 

Protease nexin 2 is identical to the secreted form of the amyloid precursor protein containing the Kunitz-type serine protease inhibitor domain (128,129), Protease nexin 2 circulates in blood stored as a platelet a-granule protein, which is secreted upon platelet activation (127). Protease nexin 2 inhibits trypsin- and chymotrypsin-like serine proteases and is also a potent inhibitor of factor Xla (126,127,128). Its location in platelets and its ability to inhibit factor Xla suggests a role in regulating blood coagulation for protease nexin 2. 

Activation of most chymotrypsin-like serine peptidases requires proteolytic processing of an inactive zymogen precursor... 

Structural Insights, Chymotrypsin A Serine Protease. Work with interactive molecular models to learn more about the structural bases of active site specificity and reactivity, and some of the ways in which active site residues can be identified. 

In the recent studies, the enzyme shows that the overall polypeptide fold of chymotrypsin-like serine protease possesses essential SI specificity determinants characteristic of elastase using the multiple isomorphous replacement (MIR) method and refined to 2.3 A resolution Fig. (5). Structure-based inhibitor modeling demonstrated that EFEa s SI specificity pocket is preferable for elastase-specific small hydrophobic PI residues, while its accommodation of long and/or bulky PI residues is also feasible if enhanced binding of the substrate and induced fit of the SI pocket are achieved [Fig. (6) shows the active sites of serine protease]. EFEa is thereby endowed with relatively broad substrate specificity, including the dual fibrinolysis. This structure is the first report of an earthworm fibrinolytic enzyme component, a serine protease originated from annelid worm [17]. 

Serine hydrolases hydrolases which have a cata-lytically active serine residue in their active center, e. g. trypsin, chymotrypsin A, B and C, thrombin and B-type carboxylic acid esterases. See Serine proteases. 

The studies now reported show that chloromethyl ketone polypeptide inhibitors bind in an antiparallel jS-pIeated sheet fashion to a length of extended backbone, Ser-125—Leu-126—Gly-127 in subtilisin, and Ser-214— Trp 215—Gly 216 in y-chymotrypsin. In each case there are the same geometric relationships of the pleated sheet to the active serine, and glycine residues are involved in j3-pleated sheet hydrogen bonding in both (see Figure 2). In the case of y-chymotrypsin these deductions were made from... 

An exceptionally reactive serine residue has been identified in a great number of hydrolase enzymes, e. g., trypsin, subtilisin, elastase, acetylcholine esterase and some lipases. These enzymes appear to hydrolyze their substrates by a mechanism analogous to that of chymotrypsin. Hydrolases such as papain, ficin and bromelain, which are distributed in plants, have a cysteine residue instead of an active serine residue in their active sites. Thus, the transient intermediates are thioesters. 

Serine proteases (e.g., trypsin, chymotrypsin, subtilisin) catalyze the hydrolysis of ester or amide substrates through an acyl-enzyme intermediate in which the hydroxyl group of the active serine side chain is acylated by the substrate (Figure 5), The reaction involves proton transfer from serine to the catalytic histidine residue, an attack on the amide carbon of the substrate, and formation of a high-energy tetrahedral intermediate. Subsequently, this intermediate breaks down, the leaving group accepts a proton from histidine and an acyl enzyme is formed which is then hydrolyzed via the reverse route. 

The active site of chymotrypsin strongly resembles that of trypsin Again there is a catalytically active serine residue which reacts with the inhibitor diisopropylfluorophosphate and whose immediate vicinity exhibits the same amino acid... 

Fig. 10.12 Sequence alignment of trypsin, chymotrypsin and thrombin (bovine). The active sites histidine, aspartic acid and serine are highlighted.

Inhibitors as well as substrates bind in this crevice between the domains. From the numerous studies of different inhibitors bound to serine pro-teinases we have chosen as an illustration the binding of a small peptide inhibitor, Ac-Pro-Ala-Pro-Tyr-COOH to a bacterial chymotrypsin (Figure 11.9). The enzyme-peptide complex was formed by adding a large excess of the substrate Ac-Pro-Ala-Pro-Tyr-CO-NHz to crystals of the enzyme. The enzyme molecules within the crystals catalyze cleavage of the terminal amide group to produce the products Ac-Pro-Ala-Pro-Tyr-COOH and NHs. The ammonium ions diffuse away, but the peptide product remains bound as an inhibitor to the active site of the enzyme. 

A closer examination of these essential residues, including the catalytic triad, reveals that they are all part of the same two loop regions in the two domains (Figure 11.10). The domains are oriented so that the ends of the two barrels that contain the Greek key crossover connection (described in Chapter 5) between p strands 3 and 4 face each other along the active site. The essential residues in the active site are in these two crossover connections and in the adjacent hairpin loops between p strands 5 and 6. Most of these essential residues are conserved between different members of the chymotrypsin superfamily. They are, of course, surrounded by other parts of the polypeptide chains, which provide minor modifications of the active site, specific for each particular serine proteinase. 

Serine proteinases such as chymotrypsin and subtilisin catalyze the cleavage of peptide bonds. Four features essential for catalysis are present in the three-dimensional structures of all serine proteinases a catalytic triad, an oxyanion binding site, a substrate specificity pocket, and a nonspecific binding site for polypeptide substrates. These four features, in a very similar arrangement, are present in both chymotrypsin and subtilisin even though they are achieved in the two enzymes in completely different ways by quite different three-dimensional structures. Chymotrypsin is built up from two p-barrel domains, whereas the subtilisin structure is of the a/p type. These two enzymes provide an example of convergent evolution where completely different loop regions, attached to different framework structures, form similar active sites. 

Figure 16.21 Structure of one subunit of the core protein of Slndbls virus. The protein has a similar fold to chymotrypsin and other serine proteases, comprising two Greek key motifs separated by an active site cleft. The C-terminus of the protein is bound in the catalytic site, making the coat protein inactive (Adapted from S. Lee et al., Structure 4 531-541, 1996.)...

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