Trypsin model

D, J Sturzebecher and WBode 1991. Geometry of Binding of the N-Alpha-Tosylated Piperidides of weffl-Amidino-Phenylalanine, Para Amidino-Phenylalanine and para-Guanidino-Phenylalanine to Thrombin and Trypsin - X-ray Crystal Structures of Their Trypsin Complexes and Modeling of their Thrombin Complexes. FEBS Letters 287 133-138. 

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. 

As these experiments with engineered mutants of trypsin prove, we still have far too little knowledge of the functional effects of single point mutations to be able to make accurate and comprehensive predictions of the properties of a point-mutant enzyme, even in the case of such well-characterized enzymes as the serine proteinases. Predictions of the properties of mutations using computer modeling are not infallible. Once produced, the mutant enzymes often exhibit properties that are entirely surprising, but they may be correspondingly informative. 

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 14.15 Stmcture of the SI fragment of chicken myosin as a Richardson diagram (a) and a space-filling model (b). The two light chains are shown in magenta and yellow. The heavy chain is colored according to three proteolytic fragments produced by trypsin a 25-kDa N-terminal domain (green) a central 50-kDa fragment (red) divided by a cleft into a 50K upper and a 50K lower domain and a 20-kDa C-terminal domain (blue) that links the myosin head to the coiled-coil tail. The 50-kDa and 20-kDa domains both bind actin, while the 25-kDa domain binds ATP. [(b) Courtesy of 1. Rayment.]...

Irreversible inhibition is probably due to the alkylation of a histidine residue.43 Chymotrypsin is selectively inactivated with no or poor inhibition of human leukocyte elastase (HLE) with a major difference the inactivation of HLE is transient.42,43 The calculated intrinsic reactivity of the coumarin derivatives, using a model of a nucleophilic reaction between the ligand and the methanol-water pair, indicates that the inhibitor potency cannot be explained solely by differences in the reactivity of the lactonic carbonyl group toward the nucleophilic attack 43 Studies on pyridyl esters of 6-(chloromethyl)-2-oxo-2//-1 -benzopyran-3-carboxylic acid (5 and 6, Fig. 11.5) and related structures having various substituents at the 6-position (7, Fig. 11.5) revealed that compounds 5 and 6 are powerful inhibitors of human leukocyte elastase and a-chymotrypsin thrombin is inhibited in some cases whereas trypsin is not inhibited.21... 

The powdery sample (e.g. a mortar) is mixed with 60 100 pi of trypsin solution (see above). Usually 5 10 mg of model fresh mortar, containing about 100 pg of proteins, is sufficient for a reliable identification. In the case of older samples (model samples aged for 9 months were tested) about 100 mg of the material was necessary. Similar amounts of samples from buildings of different age (Section 6.4.4) were needed. 

Cram and co-workers have been successful in modifying certain of their cavitands such that reactions with a bound substrate are promoted. Such systems provide a first step towards the synthesis of rudimentary enzymes (Cram, Katz, Dicker, 1984). One example of this type, involving a binding step followed by a fast acylation step, is illustrated by Figure 5.1. This sequence resembles part of the mechanism used by chymo-trypsin to cleave a peptide bond. Thus, the enzymic process entails several stages but, like the model system, begins with a binding step followed by a crucial transacylation step. 

An additional emission band near 350 nm has been observed for lima bean trypsin inhibitor (LBTI).(173) The authors discussed both the possibility of contamination by tryptophan and excited-state tyrosinate formation. Since this 350-nm emission has a tyrosine-like excitation spectrum that is slightly shifted compared to that of the major 302-nm emission, it is also possible that the tyrosine residue in a fraction of the LBTI molecules could be hydrogen bonded. This model is supported by the observations that the phenol side chain is shielded from solvent and has an anomalously high pKa. 

X.-Y. Liu, K. O. Cottrell, and T. M. Nordlund, Spectroscopy and fluorescence quenching of tyrosine in lima bean trypsin/chymotrypsin inhibitor and model peptides, Photochem. 

Another structurally simple modification involves replacement of a Lys residue with Lys(NH2) (6.83). Here, the amino group of lysine is replaced with a hydrazino group, which is less-basic by ca. 3 pKa units. This modification allows model peptides to be stabilized against endopeptidases such as trypsin and thrombin [207], Thus, the peptides Tyr-Gly-Xaa-Gly-Tyr-Ala-NH2 with Xaa = Lys or Arg were very rapidly hydrolyzed by trypsin (half-... 

Fig. 40. A classic j8 bulge the model and electron density from refined trypsin residues Ser-214, Trp-215, and Val-227. Courtesy of Chambers and Stroud.

Several proteins from different sources have been shown to maintain stability at high temperatures and NMR studies have been carried out in order to reveal their structures and/or to understand their activity. The most relevant references of a miscellany of thermostable proteins are reported in Table 3. Some of them such as bovine pancreatic trypsin inhibitor (BPTI), thermolysin and lysozyme have been widely studied as model systems in protein science. 

The three hydrolytic enzymes that have been discussed, a-chymo-trypsin, carboxypeptidase A, and lysozyme, cover a wide range of substrate types and mechanistic possibilities. Formulation of principles which might apply to enzymatic catalysis in general is difficult from such a small sampling, but certain features of the enzymatic and model reactions warrant some comment. 

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