Turkey ovomucoid inhibitor

Elastase Complex elastase/turkey ovomucoid inhibitor (enzyme /inhibitor) mimicking the natural inhibitor Solid-phase combinatorial chemistry (12 ligands) Affinity chromatography 10... 

Inspection of the X-ray structures of the third domain of turkey ovomucoid inhibitor (TOMI, a 56-residue peptidic inhibitor) with HLE and the peptidic inhibitor Ace-Ala-Pro-Val-trifluoromethylketone, 1 [Xj =210 80... 

Crystal Structure of the Complex of Human Leukocyte Elastase (PMN Elastase) and the Third Domain of the Turkey Ovomucoid Inhibitor. 

The system is particular in that the turkey ovomucoid inhibitor, TOMl (PDB access code IPPF), is an elastase inhibitor consisting of 56 residues (814 atoms), that is, characterized by a size drastically larger than the diflu-oroketone inhibitor, DFKi (PDB access code 4EST), with 70 atoms (Online Resource 1). Due to that particularity, it has been the subject of several studies [26-29] regarding their alignment using molecular similarity-based techniques. 

Fujinaga, M., et al. Crystal and molecular structures of the complex of a-chymotrypsin with its inhibitor turkey ovomucoid third domain at 1.8 A resolution. 

About 20 families of protein inhibitors of proteases have been described.488 The egg white ovomucoids comprise one family. Turkey ovomucoid is a three-domain protein whose 56-residue third domain is a potent inhibitor of most serine proteases.455 489 The 58-residue pancreatic trypsin inhibitor490 is a member of another family of small proteins. A 36-residue insect (locust) protease inhibitor is even smaller.491... 

Reduction and reoxidation have also been used to follow reactivations of biologically active proteins. It was found that an intermediate form of turkey ovomucoid (Figure 14), before complete oxidation, was actually slightly more active as an inhibitor of trypsin than was either the native protein or the completely reoxidized product. 

Figure 6. Effect of chemical modification of turkey ovomucoid on its inhibition of bovine a-chymotrypsin (by delay time assays). Abbreviations used Chy, chy-motrypsin TO, turkey ovomucoid AcTO, acetylated turkey ovomucoid AmTO, amidinated turkey ovomucoid SucTO, succinylated turkey ovomucoid 1TO, iodinated turkey ovomucoid I AcTO, iodinated and acetylated turkey ovomucoid. To a mixture of enzyme-buffer and substrate (benzoyl tyrosine ethyl ester, plus m-nitrophenol as indicator) was added the inhibitor solution within 18-25 sec and the enzyme activities recorded on a chart at 395 mfi. The weight ratio for chymotrypsin and the turkey ovomucoids was 22 15. The percent change in transmission is proportional to the amount of enzyme activity. At the time of the additions of the inhibitor, the enzyme was hydrolyzing the substrate (28).

Figure 8. Reformation of disulfides and inhibitory activities on reoxidation of reduced turkey ovomucoid, a dual inhibitor of bovine trypsin, and a-chy-motrynsin with nonoverlapping sites. The disulfides of turkey ovomucoid were reduced with mercapto-ethanol and the product purified. Reoxidation was made with protein concentrations of 60 fxg/ml in 0.006M. Tris buffer at pH 8.2 at room temperature. (Turkey ovomucoid contains eight disulfides, all of which were reduced to 16 sulfhydryls in this experiment. Of these 14 were found at the first sampling and five at the time the experiment was discontinued.) A9 Trypsin inhibition O, a-chymotrypsin inhibition , sulfhydryls per mole (33).

Fujinaga, M., Sielecki, A. R., Read, R. J., Ardelt, W., Laskowski, M., Jr., and James, M. N. (1987). Crystal and molecular structures of the complex of alpha-chymotrypsin with its inhibitor turkey ovomucoid third domain at 1.8 A resolution. J. Mol. Biol. 195, 397-418. 

The distinct nature of these multiple binding sites are shown by (a) the simultaneous binding of more than one mol/mol of a given protease or of two or more different proteases (b) amino acid sequence work (69) and (c) by fragmentation of the inhibitor molecule into separate, active parts Cyanogen bromide has been used effectively to cleave the inhibitor at methionine residues to give active fragments. Examples include the Bowman-Birk soybean inhibitor (73), turkey ovomucoid (86), the three isoinhibitors of Brazilian pink beans (77), potato inhibitor Ila (60), and potato inhibitor Ilb (62) ... 

Komiyama, T., Bigler, T.L., Yoshida, N., Noda, K. Laskowski, M. (1991). Replacement of Pi Leul8 by Glul8 in the reactive site of turkey ovomucoid third domain converts it into a strong inhibitor of glu-specific Streptomyces-griseus proteinase (GLUSGP). ]. Biol. Chem., 266, 10727-30. 

M. Fujinaga, A. R. Sielecki, R. J. Read, W. Ardelt, M. Laskowski, and M. N. G. James, /. MoL Biol., 195, 397 (1987). Crystal and Molecular Structures of the Conmiexed of a-Chymotrypsin with Its Inhibitor Turkey Ovomucoid Third Domain at 1.8 Angstroms Resolution. 

The method was tested by attempting to reproduce the bound conformations of two enzyme-inhibitor complexes Streptomyces griseus proteinase B (SGPB) complexed with the ovomucoid inhibitor from turkey (OMTKY3), and dihydrofolate reductase (DHFR) complexed with methotrexate (MTX). The... 

---