Other Serine Proteases

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). 

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.)...

Until recently, the catalytic role of Asp ° in trypsin and the other serine proteases had been surmised on the basis of its proximity to His in structures obtained from X-ray diffraction studies, but it had never been demonstrated with certainty in physical or chemical studies. As can be seen in Figure 16.17, Asp ° is buried at the active site and is normally inaccessible to chemical modifying reagents. In 1987, however, Charles Craik, William Rutter, and their colleagues used site-directed mutagenesis (see Chapter 13) to prepare a mutant trypsin with an asparagine in place of Asp °. This mutant trypsin possessed a hydrolytic activity with ester substrates only 1/10,000 that of native trypsin, demonstrating that Asp ° is indeed essential for catalysis and that its ability to immobilize and orient His is crucial to the function of the catalytic triad. 

Thus, the use of subzero temperatures in cryosolvents has allowed a series of intermediates in the elastase reaction to be identified, characterized, and stabilized. Temporal resolution of the significant steps in this reaction was difficult to achieve by any other method. Analogous results with other serine proteases suggest that these results are general for this class of enzymes. 

Cycloamyloses have been studied extensively as models of a-chymotrypsin and other serine proteases. Chymotypsin works via a double displacement pathway in which the hydroxyl group of serine-195 acts as a nucleophile. This is shown in Scheme I. In nature, the substrate is an amide. Synthetic esters are also used in model studies and routine assays. Nitrophenyl and... 

In chymotrypsin and other serine proteases the imidazole moiety of histidine acts as a general base not as a nucleophile as is probably the case in the catalysis of activated phenyl ester hydrolysis by (26). With this idea in mind, Kiefer et al. 40) studied the hydrolysis of 4-nitrocatechol sulfate in the presence of (26) since aryl sulfatase, the corresponding enzyme, has imidazole at the active center. Dramatic results were obtained. The substrate, nitrocatechol sulfate, is very stable in water at room temperature. Even the presence of 2M imidazole does not produce detectable hydrolysis. In contrast (26) cleaves the substrate at 20°C. Michaelis-Menten kinetics were obtained the second-order rate constant for catalysis by (26) is 10 times... 

Many secreted proteins, as well as smaller peptide hormones, are acted upon in the endoplasmic reticulum by tryptases and other serine proteases. They often cut between pairs of basic residues such as KK, KR, or RR.214-216 A substilisin-like protease cleaves adjacent to methionine.217 Other classes of proteases (e.g., zinc-dependent carboxypeptidases) also participate in this processing. Serine carboxypeptidases are involved in processing human prohormones.218 Among the serine carboxypeptidases of known structure is one from wheat219 and carboxypeptidase Y, a vacuolar enzyme from yeast.220 Like the pancreatic metallocarboxypeptidases discussed in Section 4, these enzymes remove one amino acid at a time, a property that has made carboxypeptidases valuable reagents for determination of amino acid sequences. Carboxypeptidases may also be used for modification of proteins by removal of one or a few amino acids from the ends. 

The value of /ccat is also pH dependent and falls off at low pH around a pKa value of from 6-7 depending upon the substrate. However, no higher pKa affects /ccat in the experimentally accessible range. These results provide the basis for believing that an unprotonated His 57 is needed in the ES complex for catalysis to occur. Similar conclusions have been reached for other serine proteases. 

The resonances of protons in hydrogen bonds may be shifted downfield to such an extent that they may be observed in H20 solutions. The proton between Asp-102 and His-57 in chymotrypsinogen, chymotrypsin, and other serine proteases has been located and its resonance found to titrate with a pKa of 7.518 (although the pKa is for the dissociation of the proton on the other nitnjjgen of the imidazole ring). ... 

The Tissue Kallikreins in the Context of Other Serine Proteases in the... 

Interactions between serine proteases are common, and substrates of serine proteases are usually other serine proteases that are activated from an inactive precursor [66]. The involvement of serine proteases in cascade pathways is well documented. One important example is the blood coagulation cascade. Blood clots are formed by a series of zymogen activations. In this enzymatic cascade, the activated form of one factor catalyzes the activation of the next factor. Very small amounts of the initial factors are sufficient to trigger the cascade because of the catalytic nature of the process. These numerous steps yield a large amplification, thus ensuring a rapid and amplified response to trauma. A similar mechanism is involved in the dissolution of blood clots. A third important example of the coordinated action of serine proteases is the intestinal digestive enzymes. The apoptosis pathway is another important example of coordinated action of other types of proteases. 

Hu JC, Zhang C, Sun X, et al. Characterization of the mouse and human PRSS17 genes, their relationship to other serine proteases, and the expression of PRSS17 in developing mouse incisors. Gene 2000 251 1-8. 

Irrimirig, P. Klar, B. Dix, D. Hydrolytic stability vs ring size in lactams implications for the development of lactam antibiotics and other serine protease inhibitors./. Med. Chem. 2000, 43, 4328—4331. 

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