Trypsin complex

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. 

The oxyanion hole geometry of three complexes is visuahzed in Figures 4.2. 4. Figure 4.2 displays the active site of trypsin complexed with a peptide inhibitor [41]. In Figure 4.3, the active site of chymotrypsin complexed with a neutral aldehyde adduct is displayed [43], and in Figure 4.4, cutinase (a lipase) with a covalently bound phosphate, a transition state analog is depicted [63]. 

Figure 4.2 Structure of the oxyanion hole of the active site of trypsin, complexed with a peptide inhibitor (PDB IPPE). The hydrogen atoms (in white) are only included when relevant for the hydrogen bonding geometry of the oxyanion hole. The dotted lines highlight the key interactions in the oxyanion hole.

These molecules are cleaved by Factor Xa suggesting they bind in a similar manner to antistasin itself. Assuming the sequence around the cleavage site occupies the FXa active site locally in a manner similar to BPTI in the BPTI trypsin complex, a modeled structure of the complex can be constructed... 

Preparation of Human Insulin. Porcine insulin can be converted to the human insulin sequence by an enzyme-catalyzed transpeptidation reaction (10,11). Under appropriate conditions trypsin acts preferentially at LysB29 rather than ArgB22 to yield a covalent des[B30]insulin/trypsin complex (acyl—enzyme intermediate). In the presence of high concentrations of organic co-solvents and the /-butyl ester of threonine, transpeptidation predominates over hydrolysis to yield the /-butyl ester of human insulin. Following appropriate purification steps and acidolytic removal of the ester, human insulin suitable for treating patients is obtained. 

The conformational flexible part found in domain II of SSI is in stark contrast with other protein proteinase inhibitors, such as BPTI. In the case of BPTI, the backbone conformation is found to be nearly identical in both the free and the bovine trypsin complex.28 The conformational rigidity of protein proteinase inhibitors has been considered for a long time to be a necessary condition to inhibit their target enzymes and to protect themselves from attack by other proteinases. It is generally recognized that the substrate-like protein proteinase inhibitors, such as BPTI, STI, and Ovomucoid domain 3,... 

Fig. 11.4. Part of the circularly polarized luminescence spectrum of the Tb3+-porcine trypsin complex showing...

R. J. Fletteeick, Macromolecular chelation as an improved mechanism of protease inhibition structure of the ecotin-trypsin complex. Embo J. 1994, 13, 1502-1507. 11... 

Further applications of n.m.r. techniques in specific enzyme systems are discussed below. E.p.r. has been used to investigate the bonding of copper(ii) in several amino-acid, peptide, and trypsin complexes, and the effects of divalent metal ions on the Raman spectrum of ATP in aqueous solution have also been reported. ... 

However, there are drawbacks to this approach, particularly the variability in the extent of 0 incorporation in the heavy -labeled peptides. This arises because of the possibility of back-exchange ( 0 replaces 0) when the peptide-trypsin complex is re-formed and subsequently hydrolyzed again. For this and other reasons this approach is not often used, since many precautions have to be taken in the acquisition and interpretation of the data (Stewart 2001). However, such labeling has been exploited (Shevchenko 1997) in experiments designed to assist in the interpretation of MS/MS spectra of peptides by distinguishing between primary fragments that contain the C-terminus (exhibit an appropriate mass shift) from N-terminal fragments (no such mass shift). 

Heating a mixed trypsin complex, which consisted of one mole each of bovine and porcine trypsin per mole of 01, in the DSC produced denaturation peaks at 79 and 85 C, close to those produced by the two 1 1 complexes (Zahnley, 1979). This result is consistent with the slow dissociation previously observed for such complexes (Zahnley, 1975). 

Reduced dissociation of mixed trypsin complexes. J. B1o1. 

Turk D, Stiirzebecher J, Bode W (1991) Geometry of binding of the Na-tosylated piperidines of ra-amidino-, p-amidino- and p-guanidino phenylalanine to thrombin and trypsin— X-ray crystal structures of their trypsin complexes and modeling of their thrombin complexes. FEES 287 133-138... 

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