Serine proteinases stabilization

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. 

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. 

The extreme stability of the acyl enzyme of serine proteinase at low temperature can be exploited, as shown below, in X-ray studies. 

Brazzein is another small sweet-tasting protein whose solution structure has been recently solved by NMR. Brazzein tastes 2000 times sweeter than sucrose on a weight basis and is exceptionally thermostable. As indicated by NMR, the structure of this 54 residue, single-chain polypeptide does not change between 32 and 82 °C and retains its sweetness after incubation at 98 °C for two hours.Brazzein contains one a-helix and three strands of antiparallel jd-sheet stabilized by four intramolecular disulphide bonds. It has been proposed that the disulphide bonds could be responsible for the thermostability of brazzein by forming a compact structure at the tertiary level.The structure of brazzein does not resemble that of the other two sweet proteins with known structures, monellin and thaumatin, whereas sequence alignment and structural prediction indicate that brazzein shares the fold of a newly identified family of serine proteinase inhibitors. 

The most important reaction in blood clotting is the conversion, catalyzed by thrombin, of the soluble plasma protein fibrinogen (factor 1) into polymeric fibrin, which is deposited as a fibrous network in the primary thrombus. Thrombin (factor 11a) is a serine proteinase (see p. 176) that cleaves small peptides from fibrinogen. This exposes binding sites that spontaneously allow the fibrin molecules to aggregate into polymers. Subsequent covalent cross-linking of fibrin by a transglutaminase (factor Xlll) further stabilizes the thrombus. 

Members of both structural families of serine proteinases, the trypsinlike and the subtilisin-like, have been found to bind Ca " (references in Tables 1 and II). The role of Ca " in all of these proteolytic enzymes appears to be one of stabilization of structure and/or maintenance of... 

Transition state analogues are essentially stable molecules which resemble, in geometry and in charge distribution, metastable intermediates of the enzymic reaction. The actual transition state of the reaction will be close in structure to the metastable intermediate, and will quite likely vary slightly between different substrates accepted by the same enzyme. There will not be a unique transition state for all transformations catalysed by one particular enzyme, neither of course will there be a unique transition state for different enzymes catalysing the hydrolysis of peptide links in a protein. There will nevertheless be some similarities in mechanism, and so structures containing a tetrahedral centre have been designed to inhibit a variety of proteinases, where a tetrahedral intermediate is always presumed. Differences exist in the pathway to, and breakdown of, the tetrahedral intermediate, and its stabilization, between thiol and serine proteinases, zinc proteinases, and aspartic proteinases. 

Fig. 10. Postulated mechanism of hydrolysis of a peptide substrate by an aspartic proteinase. Stabilization of the tetrahedral intermediate depends heavily on hydrogen bonding interaction with serine and threonine residues (not shown). Reprinted with permission from James et al. (1992). Copyright 1992 American Chemical Society.

A reaction looked at earlier simulates borate inhibition of serine proteinases.33 Resorufin acetate (234) is proposed as an attractive substrate to use with chymotrypsin since the absorbance of the product is several times more intense than that formed when the more usual p-nitrophcnyl acetate is used as a substrate. The steady-state values are the same for the two substrates, which is expected if the slow deacylation step involves a common intermediate. Experiments show that the acetate can bind to chymotrypsin other than at the active site.210 Brownian dynamics simulations of the encounter kinetics between the active site of an acetylcholinesterase and a charged substrate together with ah initio quantum chemical calculations using the 3-21G set to probe the transformation of the Michaelis complex into a covalently bound tetrahedral intermediate have been carried out.211 The Glu 199 residue located near the enzyme active triad boosts acetylcholinesterase activity by increasing the encounter rate due to the favourable modification of the electric field inside the enzyme and by stabilization of the TS for the first chemical step of catalysis.211... 

Circiunstantial support for this mechanism was supplied by the fact that A-tosyl-Phe-CMK, a specific inhibitor of chymotrypsin, did not react with anhydrochymotrypsin [104]. Although both X-ray crystallographic and NMR studies supported the alkylated hemiketal as the structure of the inhibited enzyme, those studies did not prove whether alkylation or hemiketal formation oecurred first [105, 98]. Carbon-13 NMR studies were also used to determine the pKa (7.88-8.1) of the hemiketal hydroxyl and this finding provided the first evidence that serine proteinases could stabilize the ionized form of the alkylated hemiketal, via hydrogen bonds in the oxyanion hole [106,107]. A series of more recent papers has confirmed that hemiketal formation precedes the alkylation step and has shown that the initial, reversible part of the interaction is made up of two discrete stages (a) formation of a Michaelis complex, followed by (b) hemiketal formation [102, 108]. The requirement of an intermediate hemiketal may mean that chloromethyl ketone (CMK) inhibitors should be considered as transition-state [109] analogue inhibitors (see diseussion in seetion on Aldehydes). 

The peptide aldehyde series demonstrated the potential for carbonyl derivatives to be potent inhibitors of serine proteinases. The 200-fold increase in values which was found in going from an aldehyde (7-1) Table 2.7) to its corresponding methyl ketone (7-2) Table 2.7) [129] implied that the physical properties of the carbonyl group were an important factor in determining the potency of the inhibitor. As a means of increasing both the stability and potency of peptide ketone derivatives Trainor and co-workers [130, 131] and Imperiali and Abeles [132] independently prepared the first peptide trifluoromethyl ketone (TFMK) inhibitors of serine proteinases in... 

In subtilisin, a representative of the second family of serine proteinases, the relative dispositions of Asp and His are in some contrast to those described above (Fig. 5C). The plane of the carboxyl group of the catalytic Asp is now rotated with respect to the imidazole in a direction opposite to the one observed in RmL and trypsin 081 (which accepts an H-bond from N81 of the catalytic His) is now stabilized by an additional H-bond donated by the main-chain amide of Thr-33. 

The contribution of Asp to maintaining His in such an orientation that can accept a proton from Ser is considered to be of minor importance since the electrostatic contribution to the difference in free energy between native and mutant serine proteinases accounts for most of the calculated effects of the mutations (Asp to Asn in trypsin, Asp to Ala in subtilisin). The other stabilizing effect is of the oxyanion hole on the negatively charged TI, which is also electrostatic in nature. 

TABLE II. Thermal Stabilities of Selected Protein Inhibitors of Serine Proteinases ... 

TABLE III. Thermal Stabilization of Serine Proteinases Resulting from Association with Multi-subunit Inhibitors. 

Caldolysin is the trivial name of the serine proteinase from T. aquaticus strain T351 [284]. This enzyme did not have detectable esterase aetivity and hydrolysis of small peptides of less than four amino acids was not observed. The enzyme was highly stable in the presenee of ealcium ions. Caldolysin bound six calcium ions per molecule of enzyme, there being both high and low affinity binding sites [285]. Stability of apocaldolysin (i.e. caldolysin treated with EDTA to remove all calcium ions) was restored upon incubation with either calcium or lanthanide ions, the latter giving a lanthanide-caldolysin complex more stable than the native enzyme. Strontium ions were the only other divalent metal ions tested that could restore more than 50% activity. 

However, a PrPp forces the pathogenic conformation on a PrPn. The stability towards serine proteinase K from the fungus Tritirachium album is used to differentiate between PrPp and PrPn. Serine proteinase K, which attacks the carboxyl side of hydrophobic amino acids, largely hydrolyzes PrPn while a characteristic peptide (M 27-30 kDa) is released from PrPp. This marker can be identified using the sandwich ELISA (cf. 2.6.3). 

B. Ad th and L Polgftr. Transition-state stabilization at thcoxyamon binding sites vt serine and thiol proteinase hydrolyses of thiono and oxygen eaten. Biochemistry 22 117(1983). 

Serine proteases, released from immune cell granules, process cytokines and growth factors that control multiple cellular process [56], Proteinase 3, cathepsin G, and elastase all cleave membrane-bound TNF-o, IL-1, and IL-18, and activate epidermal growth factor receptor (EGFR) and toll-like receptor-4 (TLR-4). These actions inhibit growth and lead to apoptosis with transcriptional nuclear factor kB (NF-kB) inactivation. Bik suppresses release of TNF-o, IL-1, and IL-18 and prevents EGFR and TLR-4 activation. Activation of NF-kB is a mediator of cell proliferation, whereas inhibition of NF-/. B leads to apoptosis [82]. Overall, Bik inhibition of immune cell serine proteases increases cell proliferation and stability. 

The L-aminopeptidase is sensitive to various classes of proteinase inhibitors. Strong inhibition of the enzyme is observed by treatment with the thiol reagents / -chloromercu-ribenzoate (pCMB) and iodoacetamide. The inhibition by pCMB can be reversed by subsequent treatment with dithiothreitol. In addition, the enzyme is inhibited by the metalchelating compounds EDTA and o-phenanthroline and the serine protease inhibitors phenylmethylsulfonyl fluoride and diisopropylfluorophosphate. These phenomena point to an essential serine or cysteine residue in the active site furthermore, divalent cations seem to be involved in the catalytic mechanism and/or are important for the stability of the enzyme. 

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