Trypsin inhibitor, residual

Fig. 55. Two very similar 5-residue turns with a single a-helical hydrogen bond (a) pancreatic trypsin inhibitor residues 24-28, stabilized by the side chain of Asn-24 (b) prealbumin residues 18-22, stabilized by Asp-18.

The presented algorithm was applied to 4 proteins (lysozyme, ribonuclease A, ovomucid and bovine pancreatic trypsin inhibitor) containing 51 titratable residues with experimentally known pKaS [32, 33]. Fig. 2 shows the correlation between the experimental and calculated pKaS. The linear correlation coefficient is r = 0.952 the slope of the line is A = 1.028 and the intercept is B = -0.104. This shows that the overall agreement between the experimental and predicted pKaS is good. 

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. 

So far cyclotides have been discovered in plants from the Violaceae (violet), Rubiaceae (coffee), and Cucurbitaceae (cucumber) families " and have been divided mainly into two structural subfamilies called the Mobius and bracelet cyclotides. These two cyclotide subfamilies are distinguished by the presence of a ar-proline residue in loop 5 for the Mobius subfamily. ° ° On the basis of their trypsin inhibitory activity, the two cyclotides MCoTi-I and MCoTi-II from the seeds of the tropical vine Momordica cochmchinensu form a third subfamily, referred to as the trypsin inhibitor subfamily of cyclotides. No other cyclotides have this activity. 

The peptides discussed so far are defined by a common genetic pattern or architectural feature, such as their sequence or disulfide bond pattern. In this section we discuss peptides that share a common mode of action but may arise from different peptide families. Proteinase inhibitors (Pis) come in an astounding range of sizes, from the smallest gene-encoded cyclic peptide known to date, sunflower trypsin inhibitor 1 (SFTI-1), ° a 14-residue cyclic peptide with a single disulfide bond, to squash inhibitors that are approximately 30 residues in size and feature the cystine knot motif, to 53-residue Pis found in Nicotiana... 

Despite their lack of stabilizing disulfide bridges Potl inhibitors feature a common, stable fold. The N-terminus is coiled, although in some structures a small /3-strand has been identified. After a turn the structure adopts an a-helical structure, followed by a turn and an other /3-strand. The sequence then features an extended turn or loop motif that contains the reactive site of the inhibitor before it proceeds with a /3-strand running almost parallel to the /3-strand after the a-helix. After another turn and coiled motif a short /3-strand antiparallel to the other /3-strands precedes the coiled C-terminus. Usually the N-terminal residue in the reactive site is an acidic residue followed by an aromatic amino acid, that is, tyrosine or phenylalanine. Figure 11 shows the complex of chymotrypsin inhibitor (Cl) 2 with subtilisin, the hexamer of Cl 2 from H. vulgare and a structural comparison with a trypsin inhibitor from Linum usitatissimum ... 

Trypsin inhibitors in cucumber were first found in a study by Walker-Simmons et /. " after wounding of leaves and treatment with proteinase inhibitor-inducing factor (PIIF). The amino acid sequence of two inhibitors isolated from Cucurhita maxima (winter squash) were determined by Wilusz et at The peptides named ITD I and ITD 111 each comprised a 29-residue sequence with six cysteine residues. The only difference between the two peptides is in position 9, which is lysine in ITD I and glutamic acid in ITD III. The reactive site is located at the peptide bond between Arg5 and Ile6. Owing to their discovery and distribution in Cucurbitaceae the inhibitor family has been named squash inhibitors. Since the initial discoveries many other members of the squash family have been found. 

Contacts with the catalytic residues, in combination with hydrophobic interactions, are also observed in the complex of an insect a-amylase with the Ragi bifunctional a-amylase/trypsin inhibitor (RBI) [174]. Conversely, the mechanism of inhibition of barley a-amylase by the barley a-amylase/subtilisin inhibitor (BASI) did not involve direct contact between inhibitor residues and the catalytic site [175]. The inhibitor sterically blocks the catalytic site, but does not extend into it. A cavity is created, which is occupied by a calcium ion coordinated by water-mediated interactions with the catalytic residues. 

Most protease inhibitors act by mechanisms similar to that of the pancreatic trypsin inhibitor. They are very slow substrates with a reactive loop that carries suitable Pj, P2, and P/ residues that meet the specificity requirements of the enzyme. Additional noncovalent interactions prevent dissociation and make the energy barrier for hydrolysis so high that the reaction is extremely slow.488 494 495... 

Aprotinin is a polypeptide consisting of 58 amino acid residues derived from bovine lung tissues and shows inhibitory activity toward various proteolytic enzymes including chymo-trypsin, kallikrein, plasmin, and trypsin. It was also one of the first enzyme inhibitors used as an auxiliary agent for oral (poly)peptide administration. The co-administration of aprotinin led to an increased bioavailability of peptide and protein drugs [5,44,45], The Bowman-Birk inhibitor (71 amino acids, 8 kDa) and the Kunitz trypsin inhibitor (184 amino acids, 21 kDa) belong to the soybean trypsin inhibitors. Both are known to inhibit trypsin, chymotrypsin, and elastase, whereas carboxypeptidase A and B cannot be inhibited [7,46],... 

One of the hallmarks of OBPs is the six cysteine (six half cystines) residues, but this criterion alone is not sufficient to classify a certain protein as olfactory protein. It is important to demonstrate that an OBP is expressed only (or predominantly) in olfactory tissues. Evidence for their ability to bind odorants is also desirable, but not sine qua non. One of these criteria alone would not be enough to define a given protein as an OBP. For example, bovine serum albumin (BSA) binds to insect pheromones (Leal, unpublished data) and yet it is not an OBP because it does not occur in olfactory tissues in the first place. Conversely, a protein specific to antennae is not necessarily an OBP. There are other proteins that may be expressed in antennae but not in control tissues. Non-OBPs specific to insect antennae have been previously detected (Ishida and Leal, unpublished data). Also, a g lu tath i o n e -. S -1 ra n s I e ra s e has been reported to be expressed specifically in antennae of M. sexta (Rogers et al., 1999). Likewise, the six-cysteine criterion should not be misleadingly used. Insulin and bovine pancreatic trypsin inhibitor, for example, have six cysteines in three disulfide bridges and yet they are not odorant-binding proteins. Also, mammalian and insect defensins have six well-conserved cysteine residues. 

D-domain, which contains up to 30 cysteines and up to 400 amino acid residues, shows significant sequence identity with the other D-domains, especially the cysteine, glycine, and proline residues. Further analysis of the D-domain sequences has defined a potential domain, known as the trypsin inhibitor-like cysteine-rich domain (TIL) within each D-domain sequence (Lang et al. 2004). The NH2-terminal D-domains are involved in formation of mucin disulfide-linked oligomers/multimers (Perez-Vilar and Hill 1999 Perez-Vilar and Mabolo 2007 see Section 2.3.2). 

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