Chymotrypsin serine protease family

Proteins can be classed into groups based on their overall 3-D shapes, known as protein folds (O Figure 22-la). In general, proteins that have similar functions have similar folds. This means that if you are the proud parent of an unknown protein whose structure is solved, it may be possible to make educated guesses as to the function of the protein based on its overall fold. There are a number of well-known exceptions to this [notably, the serine protease family, subtilisin and trypsin/chymotrypsin (Hartley, 1979)], but the... 

Trypsin, chymotrypsin, and elastase—three members of the serine protease family—catalyze the hydrolysis of proteins at internal peptide bonds adjacent to different types of amino acids. Trypsin prefers lysine or arginine residues chymotrypsin, aromatic side chains and elastase, small, nonpolar residues. Carboxypeptidases A and B, which are not serine proteases, cut the peptide bond at the carboxyl-terminal end of the chain. Carboxypeptidase A preferentially removes aromatic residues carboxypeptidase B, basic residues. (Illustration copyright by Irving Geis. Reprinted by permission.)... 

The sequence of a protein of interest can be compared with all other known sequences to ascertain whether significant similarities exist. Does this protein belong to one of the establishedfamiliesl A search for kinship between a newly sequenced protein and the thousands of previously sequenced ones takes only a few seconds on a personal computer (Section 7.2). If the newly isolated protein is a member of one of the established classes of protein, we can begin to infer information about the protein s function. For instance, chymotrypsin and trypsin are members of the serine protease family, a clan of proteolytic enzymes that have a common catalytic mechanism based on a reactive serine residue (Section 9.1.4). If the sequence of the newly isolated protein shows sequence similarity with trypsin or chymotrypsin, the result suggests that it may be a serine protease. 

Chymotrypsin is the most-studied member of the serine protease family of enzymes. The enzyme-catalysed hydrolytic reaction has been shown to occur in at least three kinetically distinguishable steps. The first of these consists of a very fast, diffusion-controlled formation of a non-covalent enzyme-substrate complex, followed by an acylation step. In the latter the acyl group of the substrate is covalently attached to a serine alcohol of the active site with the concomitant release of the amine of an amide substrate, or the alcohol of an ester substrate. In a final deacylation step the acyl-enzyme intermediate is hydrolysed by water, thus regenerating the free enzyme and releasing the carboxylic acid ... 

Comparison (or alignment) of amino acid sequences, also called homology search, often provides first-hand information on such conserved structural features and enables one to classify enzymes into families and predict the possible function of a new enzyme (86). A family of enzymes usually folds into similar 3-D structures, at least at the active site region. A typical example is the serine protease family whose members—trypsin, chymotrypsin, elastase, and subtilisin—commonly contain three active-site residues, Asp/His/Ser, which are known as the catalytic triad or charge relay system. Another example is the conserved features of catalytic domains of the highly diverse protein kinase family. In this kinase family, the ATP-binding (or phosphate-anchoring) sites present a consensus sequence motif of Gly-X-Gly-X-X-Gly (67,87). 

A beta barrel is a three-dimensional protein fold motif in which beta strands connected by loops form a barrellike structure. For example, this fold motif is found in many proteins of the immunoglobulin family and of the chymotrypsin family of serine proteases. 

The serine proteases are the most extensively studied class of enzymes. These enzymes are characterized by the presence of a unique serine amino acid. Two major evolutionary families are presented in this class. The bacterial protease subtilisin and the trypsin family, which includes the enzymes trypsin, chymotrypsin, elastase as well as thrombin, plasmin, and others involved in a diverse range of cellular functions including digestion, blood clotting, hormone production, and complement activation. The trypsin family catalyzes the reaction ... 

The actual reaction mechanism is very similar for the different members of the family, but the specificity toward the different side chain, R, differs most strikingly. For example, trypsin cleaves bonds only after positively charged Lys or Arg residues, while chymotrypsin cleaves bonds after large hydrophobic residues. The specificity of serine proteases is usually designated by labeling the residues relative to the peptide bond that is being cleaved, using the notation... 

NS3 is a 631 amino acid protein, and its first 180 amino acids encode a serine protease of the chymotrypsin family (Figure 2.2A). It has a typical chymotrypsin-family fold consisting of two jS-barrels, with catalytic triad residues at the interface. His-57 and Asp-81 are contributed by the N-terminal jS-barrel and Ser-139 from the C-terminal jS-barrel. NS3 and closely related viral proteases are significantly smaller than other members of the chymotrypsin family, and many of the loops normally found between adjacent jS-strands in trypsin proteases are truncated in NS3 [31]. Probably... 

From study of peptides formed by partial hydrolysis of the 32P-labeled chymotrypsin, the sequence of amino acids surrounding the reactive serine was established and serine 195 was identified as the residue whose side chain hydroxyl group became phosphorylated. The same sequence Gly-Asp-Ser-Gly was soon discovered around reactive serine residues in trypsin, thrombin, elastase, and in the trypsin-like cocoonase used by silkmoths to escape from their cocoons.198 We know now that these are only a few of the enzymes in a very large family of serine proteases, most of which have related active site sequences.199 200 Among these are thrombin and other enzymes of the blood-clotting cascade (Fig. 12-17), proteases of lysosomes, and secreted proteases. 

The serine proteases are a large family of proteolytic ( enzymes that use the reaction mechanism for nucleophilic catalysis outlined in equations (3) and (4), with a serine residue as the reactive nucleophile. The best known members of the family are three closely related digestive enzymes trypsin, chymotrypsin, and elastase. These enzymes are synthesized in the mammalian pancreas as inactive precursors termed zymogens. They are secreted into the small intestine, where they are activated by proteolytic cleavage in a manner discussed in chapter 9. 

The serine proteases are divided into at least two genetic families the mammalian serine proteases, such as trypsin, chymotrypsin, elastase, the enzymes of the blood clotting system, and many other proteases with specific roles in control of systems and the bacterial proteases called subtilisins (first to be isolated from Bacillus amyloliquefaciens), which are genetically unrelated to the mammalian enzymes but independently evolved the same mechanism (evolutionary convergence). 

Coombs, G. S., Rao, M. S Olson, A. J., Dawson, P. E and Madison, E. L. (1999) Revisiting catalysis by chymotrypsin family serine proteases using peptide substrates and inhibitors with unnatural main chains. J. Biol. Chem. 274, 24074-24079. 

The properties and spatial arrangement of the amino acid residues forming the active site of an enzyme will determine which molecules can bind and be substrates for that enzyme. Substrate specificity is often determined by changes in relatively few amino acids in the active site. This is clearly seen in the three digestive enzymes trypsin, chymotrypsin and elastase (see Topic C5). These three enzymes belong to a family of enzymes called the serine proteases - serine because they have a serine residue in the active site that is critically involved in catalysis and proteases because they catalyze the hydrolysis of peptide bonds in proteins. The three enzymes cleave peptide bonds in protein substrates on the carboxyl side of certain amino acid residues. 

Recently the effects of alcalase (another protease from subtilisin family), bovine pancreatic chymotrypsin, and papain (from papaya) have been evaluated on the desquamatory process.45 Alcalase (or Optimase) is an alkaline serine proteases derived from Bacillus licheniformis with... 

The mustard family (Brassicaceae) PIPs are 7 kDa proteins with 8 cysteines in highly conserved positions that form 4 disulphide linkages in a particular pattern of connectivity. The mustard PIPs are variously potent inhibitors of serine proteases such as trypsin, chymotrypsin, thrombin, plasmin and blood clotting factors Xa and Xlla [515, 520] (Table 15). 

Within each protease family, individual members will differ in their substrate specificity. Most proteases have extended substrate binding sites and will bind to and recognize several amino acid residues of a polypeptide substrate (see Figure 2). Usually one of these will be the primary binding site. For example, in the serine proteases chymotrypsin, trypsin, and elastase, the primary substrate binding site is the Si subsite... 

Phage libraries have also been used to study the substrate specificity of enzymes by finding an improved artificial substrate. Coombs et al. (69) reported the detailed assessment of specificity for a serine protease belonging to the a-chymotrypsin family, the prostate specific antigen (PSA). They used both substrate optimization by singlepoint mutations and phage display libraries. The sequence of the 14-member substrate 10.2 (70) was used to start the iterative optimization process (Fig. 10.11) in which substitution or exchange of the PI, P2, or P2 residues increased the substrate affinity... 

More than a third of all known proteolytic enzymes are serine proteases (2). The family name stems from the nucleophilic serine residue within the active site, which attacks the carbonyl moiety of the substrate peptide bond to form an acyl-enzyme intermediate. Nucleophilicity of the catalytic serine is commonly dependent on a catalytic triad of aspartic acid, histidine, and serine—commonly referred to as a charge relay system (3). First observed by Blow over 30 years ago in the structure of chymotrypsin (4), the same combination has been found in four other three-dimensional protein folds that catalyze hydrolysis of peptide bonds. Examples of these folds are observed in trypsin. 

The strategy used by the cysteine proteases is most similar to that used by the chymotrypsin family. In these enzymes, a cysteine residue, activated by a histidine residue, plays the role of the nucleophile that attacks the peptide bond (see Figure 9.18). in a manner quite analogous to that of the serine residue in serine proteases. An ideal example of these proteins is papain, an enzyme purified from the fruit of the papaya. Mammalian proteases homologous to papain have been discovered, most notably the cathepsins, proteins having a role in the immune and other systems. The cysteine-based active site arose independently at least twice in the course of evolution the caspases, enzymes that play a major role in apoptosis (Section 2.4.3). have active sites similar to that of papain, but their overall structures are unrelated. 

The protease family of enzymes has long generated considerable interest due to their role in peptide degradation and possibly peptide synthesis. Although many distinct families of serine proteases seem to exist, the two best studied ones are chymotrypsin and subtilisin. Several MD studies have been reported on serine proteases in aqueous media " but only little is known about the dynamics of serine proteases in non-aqueous solvents. ... 

Proteins are hydrolyzed very slowly with storage in water at neutral pH. However, addition of proteases can increase the rate of hydrolysis about 10 billion times over the spontaneous rate. The chymotrypsin mechanism depicted in Figure 2.53 is shared by trypsin and elastase. These three proteases are members of a family called the serine proteases (named after Ser 195), Carboxypeptidase Aand pepsin catalyze peptide hydrolysis by different mechanisms and are not part of this family. 

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