In serine proteinases

Naray-Szab6, G. 1983. Unusually Large Electrostatic Field Effect of the Buried Aspartate in Serine Proteinases Source of Catalytic Power. Int. J. Quant. Chem. 23, 723. 

A. Warshel, G. Naray-Szabo, F. Sussman, J. K. Hwang, How Do Serine Proteases Really Work , Biochemistry 1989, 28, 3629-3637 G. Naray-Szabo, Electrostatic Effects on Catalytical Rate Enhancements in Serine Proteinases , Int. J. Quantum Chem. 1982, 22, 575-582. 

The proteolytic enzymes are classified into endopeptidases and exopeptidases, according to their site of attack in the substrate molecule. The endopeptidases or proteinases cleave peptide bonds inside peptide chains. They recognize and bind to short sections of the substrate s sequence, and then hydrolyze bonds between particular amino acid residues in a relatively specific way (see p. 94). The proteinases are classified according to their reaction mechanism. In serine proteinases, for example (see C), a serine residue in the enzyme is important for catalysis, while in cysteine proteinases, it is a cysteine residue, and so on. 

Sumi,H. and Ulstrup, J. (1989) Dynamics of protein conformation transition in enzyme catalysis with special attention to proton transfers in serine proteinase, Biochem. Biophys. Acta 955, 26-42. 

The structures of hepatitis A viral 3C proteinases complexed with tetrapeptidyl-based methyl ketone inhibitors were shown to have an episulfide cation embedded in them. The authors concluded that the mechanism of inactivation of 3G peptidases by methyl ketone inhibitors is different than those operating in serine proteinases or in papain-like cysteine peptidases <2006MI673>. 

Until recently relatively little was known about the molecular basis of lipid hydrolysis. The first amino acid sequence of a triacylglycerol lipase was given by De Caro et al. (1981). As more sequence data became available, it was noted that lipases and esterases share a short consensus sequence, G-X-S-X-G (Boel et al., 1988 Datta et al., 1988 Antonian, 1988 Brenner, 1988). The role of the invariant serine at the center of this sequence was debated (Maraganore and Heinrikson, 1986). Some authors speculated about a lipid recognition site, others compared this pentapeptide to the sequence around the nucleophilic serine in serine proteinases. 

In RmL the nucleophilic triad consists of Ser-144, Asp-203, and His-257, whereas in hPL the analogous amino acids are Ser-153, Asp-177, and His-264. All hydrogen bonds identified in this system are analogous to those observed in serine proteinases Ne2 of the histidine is bonded to the serine hydroxyl, and N31 is H-bonded to the aspartic acid (Fig. 4). Further details of the H-bonding stereochemistry are given in Table II. 

J. L. Markley and I. B. Ibanez (1978), Zymogen activation in serine proteinases. Proton magnetic resonance pH titration studies of the two histidines of bovine chymotrypsinogen A and chymotrypsin Aa. Biochemistry 17, 4627-4639. 

Figure 2.19 Organization of polypeptide chains into domains. Small protein molecules like the epidermal growth factor, EGF, comprise only one domain. Others, like the serine proteinase chymotrypsin, are arranged in two domains that are required to form a functional unit (see Chapter 11). Many of the proteins that are involved in blood coagulation and fibrinolysis, such as urokinase, factor IX, and plasminogen, have long polypeptide chains that comprise different combinations of domains homologous to EGF and serine proteinases and, in addition, calcium-binding domains and Kringle domains.

Serine proteinase domains that are homologous to chymotrypsin, which has about 245 amino acids arranged in two domains. 

Figure 6.23 Schematic diagram illustrating the active site loop regions (red) in three forms of the serpins. (a) In the active form the loop protrudes from the main part of the molecuie poised to interact with the active site of a serine proteinase. The first few residues of the ioop form a short p strand inserted between ps and pis of sheet A. (h) As a result of inhibiting proteases, the serpin molecules are cleaved at the tip of the active site ioop region, in the cleaved form the N-terminal part of the loop inserts itself between p strands 5 and 15 and forms a long p strand (red) in the middie of the p sheet, (c) In the most stable form, the latent form, which is inactive, the N-terminai part of the ioop forms an inserted p strand as in the cleaved form and the remaining residues form a ioop at the other end of the p sheet. (Adapted from R.W. Carreii et ai., Structure 2 257-270, 1994.)...

In this chapter we shall illustrate some fundamental aspects of enzyme catalysis using as an example the serine proteinases, a group of enzymes that hydrolyze peptide bonds in proteins. We also examine how the transition state is stabilized in this particular case. 

The serine proteinases have been very extensively studied, both by kinetic methods in solution and by x-ray structural studies to high resolution. From all these studies the following reaction mechanism has emerged. 

Figure 11.6 A schematic view of the presumed binding mode of the tetrahedral transition state intermediate for the deacylation step. The four essential features of the serine proteinases are highlighted in yellow the catalytic triad, the oxyanion hole, the specificity pocket, and the unspecific main-chain substrate binding.

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. 

The serine proteinases all have the same substrate, namely, polypeptide chains of proteins. However, different members of the family preferentially cleave polypeptide chains at sites adjacent to different amino acid residues. The structural basis for this preference lies in the side chains that line the substrate specificity pocket in the different enzymes. 

As these experiments with engineered mutants of trypsin prove, we still have far too little knowledge of the functional effects of single point mutations to be able to make accurate and comprehensive predictions of the properties of a point-mutant enzyme, even in the case of such well-characterized enzymes as the serine proteinases. Predictions of the properties of mutations using computer modeling are not infallible. Once produced, the mutant enzymes often exhibit properties that are entirely surprising, but they may be correspondingly informative. 

Subtilisins are a group of serine proteinases that are produced by different species of bacilli. These enzymes are of considerable commercial interest because they are added to the detergents in washing powder to facilitate removal of proteinaceous stains. Numerous attempts have therefore recently been made to change by protein engineering such properties of the subtilisin molecule as its thermal stability, pH optimum, and specificity. In fact, in 1988 subtilisin mutants were the subject of the first US patent granted for an engineered protein. 

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. 

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