Substrate thiol proteases

A straightforward approach is to hunt for short polypeptides that meet the specificity requirement of an enzyme but which, because of peculiarities of the sequence, are acted upon very slowly. Such a peptide may contain unusual or chemically modified amino acids. For example, the peptide Thr-Pro-nVal-NMeLeu-Tyr-Thr (nVal=norvaline NMeLeu = N-methylleucine) is a very slow elastase substrate whose binding can be studied by X-ray diffraction and NMR spectroscopy.6 Thiol proteases are inhibited by succinyl-Gln-Val-Val-Ala-Ala-p-nitroanilide, which includes a sequence common to a number of naturally occurring peptide inhibitors called cystatins.f They are found in various animal tissues where they inhibit cysteine proteases. 

All proteolytic enzymes described are fairly non-specific serine endoproteases, cleaving peptide chains preferentially at the carboxyl side of hydrophobic amino acid residues. The enzymes convert their substrates into small, readily soluble fragments which can be removed easily from fabrics. Only serine protease can be used in detergent formulations, as thiol proteases such as papain would be oxidized by the bleaching agents, acidic proteases are not active at common laundry conditions, and metalloproteases such as thermolysin would lose their metal cofactors because of complexation with the water-softening agents or hydroxyl ions. 

In general the thiol proteases catalyze the hydrolysis of a variety of peptide, ester, and amide bonds of synthetic substrates. Employing the general formula R —NH—CHR—CO—X, cleavage of the —CO—X— bond has been demonstrated when R represents the side chain of glycine, threonine, methionine, lysine, arginine, citrulline, leucine, and tyrosine. 

The overall reaction pathway for the catalytic activity of the thiol proteases is best described by the scheme shown in Figure 12. This mechanism shows the formation of an enzyme-substrate complex which results in the acylation of the enzyme (to form a thiol ester) and its subsequent deacylation, the overall reaction leading to a regeneration of the enzyme, and the elimination of the products of hydrolysis. 

One of the key intermediates shown in this reaction scheme is the formation of a tetrahedral adduct during acylation and deacylation (84). Additional support for the formation of a tetrahedral intermedite comes from the observation already referred to— that aldehydes may act as potent inhibitors of papain. Westerik and Wolfenden (65) attribute the inhibitory eflFect of aldehydes to the formation of a stable thiol adduct (thiohemiacetal) analogous to the tetrahedral intermediate produced when papain acts on a substrate. This relationship is depicted in Figure 14. When the complete picture for the mechanism of catalysis by the thiol proteases finally emerges, it will no doubt be similar to the mechanism of action of the serine proteinases. 

Like the serpins, AMG is a proteinase inhibitor. It is unlike the serpins in many aspects, however. First, it is a very large molecule, with a molecular mass of -725 kDa. As a result, only very small amounts diffuse out of the plasma space. Second, it acts as a substrate for proteases but does not block their active sites instead, it enfolds the still-active proteases to block access of proteins but not small substrates. Third, it inhibits many different classes of proteinases, mcluding those with serine, cysteine, and metal ions in their proteolytic sites. Fourth, it is structurally related to pregnancy zone protein and to the complement components C3, C4, and C5 rather than to the serpins. Like these proteins, it contains an intrachain thiol ester bond that is necessary for activity and the breaking of which results in a conformational change of the peptide chain. 

Alternatively, diazomethylketone substrate derivatives can be efficiently used as active-site-directed inhibitors of thiol proteases. For instance, the carbobenzoxyphenylalanine analogue reacts stoichiometrically at the active center cysteine residue of papain. 

The group of cysteine endopeptidases (also called sulfhydryl proteases or thiol proteases) include the higher plant enzymes papain (EC 3.4.22.2) and ficin (EC 3.4.22.3), but also numerous microbial proteolytic enzymes such as Streptococcus cysteine proteinase (EC 3.4.22.10). The enzymes have a rather broad substrate specificity, and specifically recognise aromatic substituents. The specificity is for the second amino acid from the peptide bond to be cleaved. 

An enzyme reaction intermediate (Enz—O—C(0)R or Enz—S—C(O)R), formed by a carboxyl group transfer (e.g., from a peptide bond or ester) to a hydroxyl or thiol group of an active-site amino acyl residue of the enzyme. Such intermediates are formed in reactions catalyzed by serine proteases transglutaminase, and formylglyci-namide ribonucleotide amidotransferase . Acyl-enzyme intermediates often can be isolated at low temperatures, low pH, or a combination of both. For acyl-seryl derivatives, deacylation at a pH value of 2 is about 10 -fold slower than at the optimal pH. A primary isotope effect can frequently be observed with a C-labeled substrate. If an amide substrate is used, it is possible that a secondary isotope effect may be observed as welF. See also Active Site Titration Serpins (Inhibitory Mechanism)... 

The cleavage mechanism of the caspases is shown schematically in Fig. 15.5. They use a typical protease mechanism with a catalytic diad for cleavage of the peptide bond. The nucleophilic thiol of an essential Cys residue forms a covalent thioacyl bond to the substrate during the catalysis. The imidazole ring of an essential histidine is also involved in catalysis and this facilitates hydrolysis of the amide bond in the sense of an acid/base catalysis. 

With regard to the use of protease in the synthetic mode, the reaction can be carried out using a kinetic or thermodynamic approach. The kinetic approach requires a serine or cysteine protease that forms an acyl-enzyme intermediate, such as trypsin (E.C. 3.4.21.4), a-chymotrypsin (E.C. 3.4.21.1), subtilisin (E.C. 3.4.21.62), or papain (E.C. 3.4.22.2), and the amino donor substrate must be activated as the ester (Scheme 19.27) or amide (not shown). Here the nucleophile R3-NH2 competes with water to form the peptide bond. Besides amines, other nucleophiles such as alcohols or thiols can be used to compete with water to form new esters or thioesters. Reaction conditions such as pH, temperature, and organic solvent modifiers are manipulated to maximize synthesis. Examples of this approach using carboxypeptidase Y (E.C. 3.4.16.5) from baker s yeast have been described.219... 

Like other proteases, cathepsins are synthesized as high molecular weight precursors that require processing for activation. Cathepsin B (CB) is a thiol-dependent protease normally found in lysosomes, and is activated by cathepsin D (CD) and matrix metahoproteinases. Activated CB can in turn activate uPA and specific metalloproteinases. Cathepsin L (CL) is similar in specificity to that of CB however, it has little activity toward small molecular substrates. Cathepsin D, like CB, is a lysosomal protease however, CD belongs to the aspartyl group of proteases. 

Thermolysin (EC 3.4.24.4) a heat-stable, zinc- and calcium-containing neutral protease, M, 37,500, from Bacillus thermoproteolyticus, with a substrate specificity similar to that of Subtilisin (see). After one hour at 80 °C, T. still has 50% original activity. This high heat stability of T. is attributed to the large number of hydrophobic regions and the presence of four bound calcium ions, which serve in place of disulfide bridges (T. contains no disulfide bridges) to maintain the compact shape of the molecule. T. is neither a thiol nor a serine enzyme. 

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