The serine proteases

Engineering Substrate Specificity. Although the serine proteases use a common catalytic mechanism, the enzymes have a wide variety of substrate specificities. For example, the natural variant subtiHsins of B. amyloliquefaciens (subtiHsin BPN J and B. licheniformis (subtiHsin Carlsberg) possess very similar stmctures and sequences where 86 of 275 amino acids are identical, but have different catalytic efficiencies, toward tetraamino acid -nitroanilide substrates (67). 

Factor II. Prothrombin is a vitamin K-dependent compound synthesized by the Hver. When prothrombin is activated it is cleaved at two sites, resulting in a two-chain molecule linked by a disulfide bond that has a molecular weight of 37,000 daltons. Thrombin is the serine protease that initiates the conversion of soluble fibrinogen into fibrin. 

The Serine Protease Mechanism in Detail Events at the Active Site... 

FIGURE 16.26 Acyl-enzyme and amino-enzyme intermediates originally proposed for aspartic proteases were modeled after the acyl-enzyme intermediate of the serine proteases. 

Steitz, T, and Shulman, R., 1982. Cry.stallographic and NMR. studies of the serine proteases. Annual Review of Biophysics and Bioengineering 4.4.4.. 

According to their genetic relationship and their biochemical mechanism of action (3-lactamases are divided into enzymes of the serine-protease type containing an active-site serine (molecular class A, C, and D enzymes) and those of the metallo-protease type (molecular class B enzymes), which contain a complex bound zinc ion. 

The nonstructural region of the precursor, harboring the viral replication machinery, is cut into its mature components in a maturation reaction in which two viral proteases (NS2-pro and NS3/4A-pro) cooperate. Site-directed mutagenesis of an other wise infectious cDNA has shown that both HCV-encoded proteases are necessary for viral infectivity, but most of the attention has so far been focused on one of them a member of the serine protease family (EC 3.4.21) located in the N-terminal region of the viral NS3 protein. 

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 considerations presented above were based on the specific assumption that the catalytic reaction of the serine proteases involves mechanism a of Fig. 7.2. However, one can argue that the relevant mechanism is mechanism b (the so-called charge-relay mechanism ). In principle the proper procedure, in case of uncertainty about the actual mechanism, is to perform the calculations for the different alternative mechanisms and to find out which of the calculated activation barriers reproduces the observed one. This procedure, however, can be used with confidence only if the calculations are sufficiently reliable. Fortunately, in many cases one can judge the feasibility of different mechanisms without fully quantitative calculations by a simple conceptual consideration based on the EVB philosophy. To see this point let us consider the feasibility of the charge-relay mechanism (mechanism b) as an alternative to mechanism a. Starting from Fig. 7.2 we note that the energetics of route b can be obtained from the difference between the activation barriers of route b and route a by... 

Fig. 3 A ribbon diagram of the HCV NS3/4A protease ICU1 (Yao et al, 1999). The serine protease domain is shown in cyan with the catalytic triad highlighted in yellow, and the helicase domain is...

Catalysis by enzymes that proceeds via a unique reaction mechanism typically occurs when the transition state intermediate forms a covalent bond with the enzyme (covalent catalysis). The catalytic mechanism of the serine protease chymotrypsin (Figure 7-7) illustrates how an enzyme utilizes covalent catalysis to provide a unique reaction pathway. 

Factor XIa in the presence of activates factor IX (55 kDa, a zymogen containing vitamin K-dependent y-carboxyglutamate [Gla] residues see Chapter 45), to the serine protease, factor IXa. This in turn cleaves an Arg-Ile bond in factor X (56 kDa) to produce the two-chain serine protease, factor Xa. This latter reaction requires the assembly of components, called the tenase... 

The outstanding inclusion ability and the carboxylic functions of host I raised the idea of co-erystallizing it with imidazole (Im) which, due to its versatile nature 114), is one of the frequently used components in enzyme active sites, generally presented by histidine. Formally, a system made of imidazole and an acid component may mimic two essential components of the so-called catalytic triad of the serine protease family of enzymes the acid function of Aspl02 and the imidazole nucleus of His57 115) (trypsin sequence numbering). The third (albeit essential) component of the triad corresponding to the alcohol function of Seri 95 was not considered in this attempt. This family of enzymes is of prime importance in metabolitic processes. 

A different strategy for measuring protease activity is based on the property of xanthene dyes to form H-type dimers (see Sect. 6.2.3) when they are in close proximity. These dimers are accompanied with a characteristic quenching of their fluorescence and, particularly for rhodamines, with a blue shift in the absorption spectrum [121, 122]. The probe D-NorFES-D designed to measure activity of elastase in HL-60 cells consists of an undecapeptide derivatized with one tetramethylrhodamine dye on each side. The sequence contains proline residues to create a bent structure and bring the two fluoro-phores in close proximity. Intact D-NorFES-D shows 90% of its fluorescence quenched plus a blue shift of the absorption spectrum. After addition of the serine protease elastase, an increase in the fluorescence and a bathochromic shift of the absorption spectrum is observed, resulting in an increase in the emission ratio [80],... 

The natural process of thrombosis functions to plug a damaged blood vessel, thus maintaining haemostasis until the damaged vessel can be repaired. Subsequent to this repair, the clot is removed via an enzymatic degradative process known as fibrinolysis. Fibrinolysis normally depends upon the serine protease plasmin, which is capable of degrading the fibrin strands present in the clot. 

Similar studies showed a positive association between HLA alleles and development of IgE antibody. Sensitized platinum workers were DR3+ and DR6- while non-sensitized workers were DR3- and DR6+ [37], The linkage between HLA phenotype and development of IgE antibody was greatest among the low exposure group. A second study showed a relationship between HLA DR3 and IgE to inhaled anhydrides [38], A study among detergent workers showed a positive association between HLA-DR4+ and IgE antibody to the serine protease derived from Bacillus licheniformis [39],... 

Figure 7. Two examples of irreversible inactivators that are not suicide substrates a) TPCK, a classic" affinity label of the serine protease chymotrypsin, b) ZFK-CH2-mesitoate, a quiescent" affinity label of the cysteine protease cathepsin B, and c) the kinetic scheme for both forms of affinity label-inactivation.

Scallan, M.J., Raj, B.K.M., Calvo, B., Garin-Chesa, P., Sanz-Moncasi, M.P., Healey, J.H., Old, L.J. and Rettig, W.J. (1994) Molecular cloning of fibroblast activation protein alpha, a member of the serine protease family selectively expressed in stromal fibroblast of epithelial cancers. Proceedings of the National Academy of Sciences ofthe United States of America, 91, 5657-5661. 

The effects of Lp(a) on the fibrinolytic system are based on the homology between plasminogen and Lp(a) (E3, E5, K4). Inactive Glu-plasminogen is converted to inactive glutamine-plasmin or inactive lysine-plasmin. Both can be converted to active lysine-plasmin, the activity of which is based on the serine protease part that splits fibrin and fibrinogen, but also factors V and Villa. In addition, Lp(a) is able to activate factor XII, factor VII, and the complement factors Cl and C3. 

A. A. Kossiakoff, S. A. Spencer, Direct Determination of the Protonation States of Aspartic Acid-102 and Histidine-57 in the Tetrahedral Intermediate of the Serine Proteases Neutron Structure of Trypsin , Biochemistry 1981, 20, 6462-6474. 

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