Trypsin substrates

Fig. 4.4 Increase of p-nitroaniline in a solution containing the trypsin substrate benzoyl-arginine-p-nitroanilide and trypsin at 37°C and phosphate buffer pH 7.6 -o- buffer only, - -0.1% chitosan, -A- 0.025% chitosan-aprotinin,...

Design of Trypsin Substrates of a New Type — Inverse Substrates.98... 

Enzymes which catalyze the hydrolysis of the unit linkage of sequential residues of oligomers or polymers determine their substrate specificity by recognizing the particular unit residue in the sequential chain as well as the direction of the chain. For example, ribonuclease cleaves the 3 -phosphate of a pyrimidine nucleotide residue but not the 5 -phosphate, and trypsin hydrolyzes peptide bonds which involve the arginine or lysine residue at the carbonyl end but not at the amino end. This is also the case for the hydrolysis of a variety of synthetic substrates and quasi-substrates (Sect. 4.1). Synthetic trypsin substrates are ester or amide derivatives in which the site-specific group (positive charge) is contained in their carbonyl portion. 

The specificity of chymotrypsin for hydrolysis of peptide bonds formed by the carbo,xyl groups of tyrosine, phenylalanine, and tryptophan has been recognized for some time (Green and Neurath, 1954 Desnuelle, 1960). Action on synthetic substrates of leucine (Goldenberg et al., 1951) and methionine (Kaufman and Neurath, 1949) also has been noted although at much slower rates than observed with the aromatic amino acid derivatives. When protein substrates or synthetic ester substrates are examined, it is evident that a variety of bonds can be hydrolyzed by chymotrypsin. Inagami and Sturtevant (1960) observed that chymotryptic hydrolysis of a-benzoyl-L-arginine ethyl ester, a typical trypsin substrate, occurred at a maximum rate which was 20% of that observed with trypsin. Several ester substrates, such as p-nitrophenylacetate (Hartley and Kilby, 1954), are also hydrolyzed. 

Figure 12.5-17. Schematic comparison of the binding of a peptide 4-gua-nidinophenyl ester and a common trypsin substrate to the active site of the enzyme according to the conventional binding model.

How does it work from the mechanistic point of view Contrary to common trypsin substrates, the acyl residues of these enzyme-substrate mimetic arrangements bind to the S -subsite of trypsin (Fig. 12.5-18). For this reason, all binding sites beyond Si are only of minor importance for the substrate mimetics. Furthermore, the acyl residues of the substrate mimetics do not reflect the specificity of the S-... 

This Highlight is part of an extraordinary story (also a cautionary tale) in the area of biocatalysis. The point of particular interest was the incredible catalytic activity claimed for so-called pepzymes - small synthetic peptides modelled to mimic the active site structures of trypsin and chymotrypsin. One was claimed to hydrolyse a simple peptide (a trypsin substrate) with efficiency comparable to that of the native enzyme. This extraordinary result provoked at least as much scepticism as excitement, and in the following months several groups tried to reproduce the results. They failed, comprehensively. [1,2] Some reasons why this failure came as no surprise were subsequently summarised by Matthews, Craik and Neurath, [3] and by Corey and Corey [4]. The background has been discussed in an Angewandte Review on Enzyme Mechanisms, Models and Mimics. [5]... 

Not only does this molecule hydrolyse the simple trypsin substrate A-tosyl-L-arginine methyl ester with kcat and Km comparable to those of the native enzyme, but it also hydro-... 

Elastase (EC 3.4.21.11) an endopeptidase specific for the Elastic (see) in animal elastic fibers. Its inactive precursor, proelastase, is formed in the vertebrate pancreas and converted in the duodenum to elastase by the action of trypsin. The natural substrate of E. is elas-tin, an insoluble protein rich in valine, leucine and isoleucine. E. attacks the peptide bond adjacent to a nonaromatic, hydrophobic amino acid. The best synthetic substrates are therefore acetyl-Ala-Ala-Ala-OCHj and benzoylalanine methyl ester. Benzoylarginine ester (a trypsin substrate), and acetyltyrosine ester (a chymotrypsin substrate) are not attacked by E. 

Enzyme Nomenclature. The number of enzymes known exceeds two thousand. A system of classification and nomenclature is required to identify them unambiguously. During the nineteenth century, it was the practice to identify enzymes by adding the suffix -in to the name of their source. Names such as papain, ftcin, trypsin, pepsin, etc, are still in use. However, this system does not give any indication of the nature of the reaction catalyzed by the enzyme or the type of substrate involved. 

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.

How would substrate preference be changed if the glycine residues in trypsin at positions 216 and 226 were changed to alanine rather than to the more bulky valine and threonine groups that are present in elastase This question was addressed by the groups of Charles Cralk, William Rutter, and Robert Fletterick in San Francisco, who have made and studied three such trypsin mutants one in which Ala is substituted for Gly at 216, one in which the same substitution is made at Gly 226, and a third containing both substitutions. 

The Asp 189-Lys mutation in trypsin causes unexpected changes in substrate specificity... 

Asp 189 at the bottom of the substrate specificity pocket interacts with Lys and Arg side chains of the substrate, and this is the basis for the preferred cleavage sites of trypsin (see Figures 11.11 and 11.12). It is almost trivial to infer, from these observations, that a replacement of Asp 189 with Lys would produce a mutant that would prefer to cleave substrates adjacent to negatively charged residues, especially Asp. On a computer display, similar Asp-Lys interactions between enzyme and substrate can be modeled within the substrate specificity pocket but reversed compared with the wild-type enzyme. 

The results of experiments in which the mutation was made were, however, a complete surprise. The Asp 189-Lys mutant was totally inactive with both Asp and Glu substrates. It was, as expected, also inactive toward Lys and Arg substrates. The mutant was, however, catalytically active with Phe and Tyr substrates, with the same low turnover number as wild-type trypsin. On the other hand, it showed a more than 5000-fold increase in kcat/f m with Leu substrates over wild type. The three-dimensional structure of this interesting mutant has not yet been determined, but the structure of a related mutant Asp 189-His shows the histidine side chain in an unexpected position, buried inside the protein. 

Mutations in the specificity pocket of trypsin, designed to change the substrate preference of the enzyme, also have drastic effects on the catalytic rate. These mutants demonstrate that the substrate specificity of an enzyme and its catalytic rate enhancement are tightly linked to each other because both are affected by the difference in binding strength between the transition state of the substrate and its normal state. 

Craik, C.S., et al. Redesigning trypsin alteration of substrate specificity. Science 228 291-297, 1985. 

Graf, L., et al. Selective alteration of substrate specificity by replacement of aspartic acid 189 with lysine in the binding pocket of trypsin. Biochemistry 26 ... 

Krieger, M., Kay, L.M., Stroud, R.M. Structure and specific binding of trypsin comparison of inhibited derivatives and a model for substrate binding. /. Mol. Biol. 83 209-230, 1974. 

FIGURE 16.19 The substrate-binding pockets of trypsin, chymotrypsin, and elastase. 

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. 

---