Chymotrypsin substrate preferences

Proteasomes of Thermoplasma contain a single type of p subunit but eukaryotic proteasomes contain subunits with at least three distinct substrate preferences.347 M9c They all appear to use the same hydrolytic mechanism but in their substrate specificities they are chymotrypsin-like, peptidylglutamyl-peptide hydrolyzing, branched chain amino acid preferring, and small neutral amino acid preferring based on the P, amino acid residue. In the spleen some of the P subunits of the proteasomes appear to have been replaced by proteins encoded by the major histocompatibility complex of the immune system (Chapter 31).347 This may alter the properties of the proteasome to favor their function in antigen processing. Proteasomes are also ATP- and ubiquitin-dependent, as discussed in Section 6. 

In terms of structure and function, the 265 proteasome is an ATP-dependent multicatalytic enzyme complex comprising one or two 195 regulatory caps and a proteolytic 205 core particle within which protein degradation occurs.18,25,26 The 205 proteasome contains three pairs of proteolytic subunits, (35, (32 and (31, for which chymotrypsin-like (CT-L), trypsin-like (T-L) and caspase-like (C-L) activities have been ascribed, respectively, based on their substrate preferences.27... 

A simultaneous regio- and enantioselective hydrolysis of dimethyl 2-methylsuc-cinate has been reported with PPL [381] with a preference for the (S)-ester and with the hydrolysis taking place at position 4 (Scheme 2.52). The residual unhydrolyzed ester was obtained with >95% e.e. but the monoacid formed (73% e.e.) had to be re-esterified and subjected to a second hydrolytic step in order to be obtained in an optically pure form. It is interesting to note that a-chymotrypsin exhibited the same enantio- but the opposite regioselectivity on this substrate, preferably hydrolyzing the ester at position 1 [382]. 

Pish silage prepared by autolysis of rainbow trout viscera waste was investigated as a substrate for the plastein reaction using pepsin (pH 5.0), papain (pH 6—7), and chymotrypsin (pH 8.0) at 37°C for 24 h (152). Precipitation with ethanol was the preferred recovery method. Concentration of the protein hydrolysate by open-pan evaporation at 60°C gave equivalent yields and color of the final plastein to those of the freeze-dried hydrolysate. 

Residue 189 is at the bottom of the specificity pocket. In trypsin the Asp residue at this position interacts with the positively charged side chains Lys or Arg of a substrate. This accounts for the preference of trypsin to cleave adjacent to these residues. In chymotrypsin there is a Ser residue at position 189, which does not interfere with the binding of the substrate. Bulky aromatic groups are therefore preferred by chymotrypsin since such side chains fill up the mainly hydrophobic specificity pocket. It has now become clear, however, from site-directed mutagenesis experiments that this simple picture does not tell the whole story. 

Figure 11.11 Schematic diagrams of the specificity pockets of chymotrypsin, trypsin and elastase, illustrating the preference for a side chain adjacent to the scisslle bond In polypeptide substrates. Chymotrypsin prefers aromatic side chains and trypsin prefers positively charged side chains that can interact with Asp 189 at the bottom of the specificity pocket. The pocket is blocked in elastase, which therefore prefers small uncharged side chains.

More specific evidence came from affinity labeling with molecules which could react with specific amino acid group sat or adjacent to the substrate site. These labels were substrate analogues and competitive inhibitors. Substituted aryl alkyl ketones were used. TV-p-toluene-sulphonyl-L-phenylalanine chloromethyl ketone (TPCK) blocked the activity of chymotrypsin. Subsequent sequence analysis identified histidine 57 as its site of binding (see Hess, 1971, p 213, The Enzymes, 3rd ed.). Trypsin, with its preference for basic rather than aromatic residues adjacent to the peptide bond, was not blocked by TPCK but was susceptible to iV-p-toluenesulphonyl-L-lysine chloromethyl ketone (TLCK) (Keil, ibid, p249). 

Various esterases exist in mammalian tissues, hydrolyzing different types of esters. They have been classified as type A, B, or C on the basis of activity toward phosphate triesters. A-esterases, which include arylesterases, are not inhibited by phosphotriesters and will metabolize them by hydrolysis. Paraoxonase is a type A esterase (an organophosphatase). B-esterases are inhibited by paraoxon and have a serine group in the active site (see chap. 7). Within this group are carboxylesterases, cholinesterases, and arylamidases. C-esterases are also not inhibited by paraoxon, and the preferred substrates are acetyl esters, hence these are acetylesterases. Carboxythioesters are also hydrolyzed by esterases. Other enzymes such as trypsin and chymotrypsin may also hydrolyze certain carboxyl esters. 

Substrate specificity. Like most other enzymes, proteases display distinct preferences for certain substrates. These are often discussed using the nomenclature of Fig. 12-14. The substrate residue contributing the carbonyl of the amide group to be cleaved is designated Pj and residues toward the N terminus as P2, P3, etc., as is shown in Fig. 12-14. Residues toward the C terminus from the peptide linkage to be cleaved are designated P P etc. Chymotrypsin acts... 

Conformational Energy Calculations of Enzyme-Substrate Interactions. I. Computation of Preferred Conformation of Some Substrates of a-Chymotrypsin. 

The proteasome has multiple active sites. Three activities, chymotrypsin-like, tryp-sin-like and PGPH, are classified by their respective substrate specificities they all differ in the preferred amino acid in the PI position adjacent to the scissile amide bond. Proteasome inhibitors can be divided into several groups based on pharmacophores (Tab. 3.1, Fig. 3.7). 

The Gommission on Biochemical Nomenclature assigns enzyme numbers to 18 serine proteases in the 1972 edition of Enzyme Nomenclature (14). Seven are listed as having a trypsin-like specificity, i,e, their specific substrates have a positively charged lysine or arginine residue at Pi. Three are listed as having a chymotrypsin-like specificity, i.e., their specific substrates have residues of tryptophan, tyrosine, phenylalanine, or leucine at Pi, i,e, residues with bulky, hydrophobic side chains. Two enzymes have elastase-like specificities. They prefer a residue with... 

In principle, the use of amino acid or peptide esters as nucleophilic components in protease-catalyzed synthesis is possible, but with a drastically decreased efficiency. However, acyl transfer to arginine or lysine alkyl esters in ice using a-chymotrypsin with regard to its strong preference for basic residues in the P/ position enabled synthesis of a N-protected tripeptide ester in high yield (Scheme 7, see Section 4.2.1.2.2). Furthermore, it was found to be the method of choice in synthesizing new potential protease substrates (for proteases with a preference for basic residues in the Pj position). Neither enzymatic synthesis at room temperature nor synthesis in organic solvents has been shown to proceed in a successful manner. 

Proteolytic enzymes, such as the serine proteases, are among the best characterized of all enzymes.They are important in digestive processes because they break down proteins. They each catalyze the same type of reaction, that is. the breaking of peptide bonds by hydrolysis. The crystal structures of several serine proteases have been determined, and the mechanism of hydrolysis is similar for each. The specificity of each enzyme is, however, different and is dictated by the nature of the side chains flanking the scissile peptide bond (the bond that is broken in catalytic mechanism. Chymotrypsin is one of the best characterized of these serine proteases. The preferred substrates of chymotrypsin have bulky aromatic side chains. The crystal structure determination of the active site of chymotrypsin, illustrated in Figure 18.12, has provided much of the information used to elucidate a plausible mechanism of action of the enzyme. In the first step of any catalyzed reaction, the enzyme and substrate form a complex, ES, the Michaelis complex. The hydrolysis of the peptide bond by chymotrypsin involves three amino acid residues,... 

In the recent studies, the enzyme shows that the overall polypeptide fold of chymotrypsin-like serine protease possesses essential SI specificity determinants characteristic of elastase using the multiple isomorphous replacement (MIR) method and refined to 2.3 A resolution Fig. (5). Structure-based inhibitor modeling demonstrated that EFEa s SI specificity pocket is preferable for elastase-specific small hydrophobic PI residues, while its accommodation of long and/or bulky PI residues is also feasible if enhanced binding of the substrate and induced fit of the SI pocket are achieved [Fig. (6) shows the active sites of serine protease]. EFEa is thereby endowed with relatively broad substrate specificity, including the dual fibrinolysis. This structure is the first report of an earthworm fibrinolytic enzyme component, a serine protease originated from annelid worm [17]. 

Previous studies have demonstrated that subtilisin has a chymotrypsin-like specificity (reviewed in 9 ), preferring to hydrolyze the peptide bond following a large hydrophobic residue. A model of substrate binding has been deduced from the three-dimensional models of peptide and protein inhibitor complexes with subtilisin (10). The model we have derived is shown in Figure 1. The enzyme can be divided in a series of subsites... 

Next to trypsin chymotrypsin is the most preferred proteolytic enzyme in sequencing. Its specificity is less absolute than that of trypsin. Primarily the bonds that follow phenylalanine, tyrosine and tryptophan are cleaved, but measurable hydrolysis takes place next to leucine and methionine residues as well. It is advisable, therefore, to determine in preliminary experiments the conditions (enzyme-substrate ratio, time, temperature) best suited for the formation of a few and well separable fragments. Occasionally also less specific enzymes, such as pepsin, papain or thermolysin find application in structure elucidation. For the hydrolysis of specific bonds new microbial proteases can be isolated. There are known prolidases and also enzymes which hydrolyze solely the bond which follows a pyroglutamyl residue and so on. 

The preference of pepsin for peptide bonds involving two aromatic or hydrophobic AA residues are previously observed by Tang (5) and with synthetic substrates by various investigators (see Chapter 8 in this volume). This is in striking contrast to chymotrypsin and... 

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