Three-dimensional structures trypsin

This is nicely illustrated by members of the chymotrypsin superfamily the enzymes chymotrypsin, trypsin, and elastase have very similar three-dimensional structures but different specificity. They preferentially cleave adjacent to bulky aromatic side chains, positively charged side chains, and small uncharged side chains, respectively. Three residues, numbers 189, 216, and 226, are responsible for these preferences (Figure 11.11). Residues 216... 

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. 

Sprang, S., et al. The three-dimensional structure of Asn ° mutant of trypsin role of Asp ° in serine protease catalysis. Science 237 905-909, 1987. 

FIGURE 6.25 The three-dimensional structure of bovine pancreatic trypsin inhibitor. 

S. Sprang, T. Standing, R. J. Fletterick, R. M. Stroud, J. Finer-Moore, N. H. Xoung, R. Hamlin, W. J. Rutter, C. S. Craik, The Three-Dimensional Structure of Asn102 Mutant of Trypsin Role of Asn102 in Serine Protease Catalysis , Science 1987, 237, 905-909. 

The three-dimensional structure of trypsin inhibitor 1 from C. maxima was determined in 1989 by X-ray crystallography in complex with bovine trypsin and by in aqueous solution. The three-dimensional... 

Associated with the problem of active-site titration is the question of the location of the active site in the three-dimensional structure of the protein. As a prelude to this investigation, a study is needed to indicate which amino acid residues in the overall peptide sequence are in the active site. The active site is defined as the location of the enzyme catalysis thus, the substrate complexes at the active site prior to the catalytic process. Addition of a substrate will, therefore, protect the enzyme against reagents, such as inhibitors, which react at the active site. Of course, the active site may include amino acid residues from distant parts of the peptide chain for example, both serine-195 and histidine-57 are in the active site of a-chymo trypsin. 

Kallikreins can be roughly divided into two categories, the classical kallikreins (hKl, hK2, and hK3) and the new kallikreins. The new kallikreins appear to be unique in their three-dimensional structure and share some features with trypsins and other features with the classical kallikreins. Comparative protein models show that the pattern of hydrophobic side-chain packing in the protein core is nearly identical in all human kallikreins, and the observed differences occur within the solvent-exposed loop segments. 

Figure 12 Three-dimensional structures of kalata B2 (a), cycloviolacin Ol (b) and MCoTI-ll (c) (PDB codes 1PT4, 1NBJ and 1IB9, respectively), shown as representatives of the Mobius, bracelet and trypsin inhibitor subfamilies of cyclotides.

Thrombin is a proteolytic enzyme and has a remarkable similarity in its overall three-dimensional structure to the digestive serine proteases, trypsin, and chymotrypsin [11-13]. Trypsin and thrombin share a common primary specificity for proteolysis next to arginine or lysine residues. Structural data of thrombin and trypsin have demonstrated strong resemblance in their substrate sites, and many small organic inhibitors are comparably active against both the enzymes [14,15]. For this reason, no or low inhibition of trypsin is viewed as a required condition for a compoimd to be a successful orally bioavailable thrombin inhibitor [16]. 

Trypsin was named more than 100 years ago. It and chymotrypsin were among the first enzymes to be crystallized, have their amino acid sequences determined, and have their three-dimensional structure outlined by x-ray diffraction. Furthermore, both enzymes hydrolyze not only proteins and peptides but a variety of synthetic esters, amides, and anhydrides whose hydrolysis rates can be measured conveniently, precisely and, in some instances, extremely rapidly. As a result, few enzymes have received more attention from those concerned with enzyme kinetics and reaction mechanisms. The techniques developed by the pioneers in these various fields have enabled other serine proteases to be characterized rapidly, and the literature on this group of enzymes has become immense. It might be concluded that knowledge of serine proteases is approaching completeness and that little remains but to fill in minor details. 

This discussion of the metalloexopeptidases has focused on the general role of these enzymes in the conversion of dietary proteins into amino acids. In particular, the apparent synergistic relationship which the pancreatic carboxypeptidases have with the major endopeptidases, trypsin, chymotrypsin, and pepsin, in order to facilitate formation of essential amino acids has been stressed. The chemical characteristics, metalloenzyme nature, and mechanistic details of a representative of each class of exopeptidase have been presented. Leucine aminopeptidase from bovine lens was shown to be subject to an unusual type of metal ion activation which may be representative of a more general situation. Carboxypeptidase A of bovine pancreas was discussed in terms of its three-dimensional structure, the implications of x-ray crystallography to mecha ... 

The NA spikes can be eluted by pronase or trypsin treatment, yielding crystallisable, enzymatically-active, and antigenically equivalent tetramers, which has allowed thdr three-dimensional structure to be determined. 

Labeyrie et al. (41) isolated a trypsin fragment of 11 kDa from S. cerevisiae flavocytochrome 62 that contained heme but was devoid of flavin and had no lactate dehydrogenase activity. The fragment, which was referred to as cytochrome 62 core, was found to have spectral properties very like those of microsomal cytochrome 65 (41). This similarity with cytochrome 65 is borne out by comparisons of amino acid sequence (42-44). The sequence similarity extends to related heme domains of sulfite oxidase (45, 46) and assimilatory nitrate reductase (47). The existence of a protein family with a common cytochrome 65 fold was suggested by Guiard and Lederer (48) and this is supported by the close similarity between the three-dimensional structures of microsomal cytochrome 65 (49) and the cytochrome domain of flavocytochrome 62 (23-25). 

Convergence may also occur when the sequence and structure of molecules are very different, but the mechanisms by which they act are similar. Serine proteases have evolved independently in bacteria (e.g. subtilisin) and vertebrates (e.g. trypsin). Despite their very different sequences and three-dimensional structures, in each the same set of three amino acids form the active site. The catalytic triads are His57, Aspl02, and Serl95 (trypsin) and Asp32, His64, and Ser221 (subtilisin) (Doolittle, 1994 A. Tramontano, personal communication). 

Much but not all of this work has dealt with proteins the three-dimensional structures of which have been determined by x ray lysozyme, ribonuclease, myoglobin, hemoglobin, cytochrome C, carboxy-peptidase, chymotrypsin, concanavalin, trypsin, elastase, and sub-tilisin. The principal nucleus has been the proton, but more recently 13 C has been studied by several groups. Other nuclei, such as 19 F, 31P, and 35Cl, have found limited application in special studies. 

The first crystallization of cytochrome 65, obtained from pig liver was reported by Raw and Coli in 1959 (10 ). Kajihara and Hagihara (129) obtained three crystalline cytochrome be preparations from rabbit liver microsomes, two from trypsin extracts, and one from Nagarse (subtilisin BPN ) extracts. The three preparations crystallized in entirely different shapes. Calf liver cytochrome bs has also been crystallized by Mathews and Strittmatter (ISS). The preparations by the latter two groups have been used for studies on the amino acid sequence and the three-dimensional structure, respectively. 

We have already seen that trypsin, chymotrypsin and thrombin have very similar 3D structures. If we now overlay the three-dimensional structures of these enzymes we find that identical amino acids are found at many positions in space, including the active site serine, hrstidine and aspartic acid residues, as shown in Figure 10.11 (colour plate section). The amino acid sequences of these proteins can be arranged into a sequence alignment as... 

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