Proteinases reactions

Olson ST, Bjork I, Sheffer R, Craig PA, Shore JD, Choay J. Role of the antithrombin-binding pentasaccharide in heparin acceleration of antithrombin-proteinase reactions. Resolution of the antithrombin conformational... 

Lawrence DA, Olson ST, Muhammad S et aL Partitioning of serpin-proteinase reactions between stable inhibition and substrate cleavage is regulated by the rate of serpin reactive center loop insertion into fl-sheet A. J Biol Chem 2000 275 5839-5844. 

The serine proteinases have been very extensively studied, both by kinetic methods in solution and by x-ray structural studies to high resolution. From all these studies the following reaction mechanism has emerged. 

Immunologic abnormahties (eg, transfusion reactions, the presence in plasma of warm and cold antibodies that lyse red blood cells, and unusual sensitivity to complement) also fall in this class, as do toxins released by various infectious agents, such as certain bacteria (eg, Clostridium). Some snakes release venoms that act to lyse the red cell membrane (eg, via the action of phospholipases or proteinases). 

Enzymatic synthesis of aliphatic polyesters was also achieved by the ringopening polymerization of cyclic diesters. Lactide was not enzymatically polymerized under mild reaction conditions however, poly(lacfic acid) with the molecular weight higher than 1 x 10" was formed using lipase BC as catalyst at higher temperatures (80-130°C). Protease (proteinase K) also induced the polymerization however, the catalytic activity was relatively low. 

Mor, A. Maillard, J. Favreau, C. Reboud-Ravaux, M. Reaction of thrombin and proteinases of the fibrinolytic system with a mechanism-based inhibitor, 3,4-dihydro-3-benzyl-6-chloromethyl-coumarin. Biochim. Biophys. Acta 1990, 1038, 158-163. 

Orlowski, M. Cardozo, C. Eleutri, A. M. Kohanski, R. Kam, C. M. Powers, J. C. Reactions of [14C]-3,4-dichloroisocoumarin with subunits of pituitary and spleen multi-catalytic proteinase complexes (proteasomes). Biochemistry 1997, 36, 13946-13953. 

The fluidity of blood is a result of the inhibition of a complex series of enzymic reactions in the coagulation cascade (see Fig. 10). When triggered either intrinsically (by contact with foreign surfaces ), or extrinsically (by tissue factors from damaged cells), inactive proenzymes (factors XII, XI, IX, and X) are transformed into activated pro-teinases (XHa, XIa, IXa, and Xa, respectively). Each proteinase catalyzes the activation of the following proenzyme in the sequence, up to formation of thrombin (Factor Ha), another proteinase that catalyzes partial... 

B4. Barrett, A. J., and Starkey, P. M., The interaction of a2-macroglobulin with proteinases. Characteristics and specificity of the reaction, and a hypothesis concerning its molecular mechanism. Biochem. J. 133,709-724 (1973). 

BINAP-Ru is effective for the diastereoselective hydrogenation of some chiral yS-keto esters (Fig. 32.13). Reaction of N-Boc-protected (S)-y-amino / -keto esters 13A catalyzed by the (R)-BINAP-Ru complex results in the syn alcohols 13B exclusively [52]. The stereocenter at the / -position is controlled by the chirality of the catalyst therefore, use of the S catalyst affords the anti isomer, as predicted. Derivatives of statine, a key component of the aspartic proteinase inhibitor pep-... 

For activity assays, proteinase solutions were made fresh daily (10 mg freeze-dried solids in 1 ml pH 10.0 phosphate buffer, 0.1 M). Two ml of the proteinase, 0.5 ml of substrate (azocasein or other proteins in pH 10 buffer), 0.3 ml of 0.1% EDTA, and deionized water were made up to a volume of 3.5 ml. Reaction tubes were incubated in a 40°C water bath for 1 hr, then the reaction was stopped by addition of 1 ml 5% TCA. After removal of the precipitated proteins by centrifugation, absorbance was read at 366 nm (for azocasein) or by the Lowry method (15) for other... 

The proteolytic enzymes are classified into endopeptidases and exopeptidases, according to their site of attack in the substrate molecule. The endopeptidases or proteinases cleave peptide bonds inside peptide chains. They recognize and bind to short sections of the substrate s sequence, and then hydrolyze bonds between particular amino acid residues in a relatively specific way (see p. 94). The proteinases are classified according to their reaction mechanism. In serine proteinases, for example (see C), a serine residue in the enzyme is important for catalysis, while in cysteine proteinases, it is a cysteine residue, and so on. 

The most important reaction in blood clotting is the conversion, catalyzed by thrombin, of the soluble plasma protein fibrinogen (factor 1) into polymeric fibrin, which is deposited as a fibrous network in the primary thrombus. Thrombin (factor 11a) is a serine proteinase (see p. 176) that cleaves small peptides from fibrinogen. This exposes binding sites that spontaneously allow the fibrin molecules to aggregate into polymers. Subsequent covalent cross-linking of fibrin by a transglutaminase (factor Xlll) further stabilizes the thrombus. 

Normally, thrombin is present in the blood as an inactive proenzyme (see p. 270). Prothrombin is activated in two different ways, both of which represent cascades of enzymatic reactions in which inactive proenzymes (zymogens, symbol circle) are proteolytically converted into active proteinases (symbol sector of a circle). The proteinases activate the next proenzyme in turn, and so on. Several steps in the cascade require additional protein factors (factors 111, Va and Villa) as well as anionic phospholipids (PL see below) and Ca "" ions. Both pathways are activated by injuries to the vessel wall. 

This enzyme [EC 3.4.21.53], also known as endopepti-dase La, ATP-dependent serine proteinase, and ATP-dependent protease La, catalyzes the hydrolysis of peptide bonds in large proteins (for example, globin, casein, and denaturated serum albumin) in the presence of ATP (which is hydrolyzed to ADP and orthophosphate). Vanadate ion inhibits both reactions. A similar enzyme occurs in animal mitochondria. Protease La belongs to the peptidase family S16. 

Fig. 8. Morphological changes of apoptotic eosinophils induced by dexamethasone (Z2). After eosinophils were treated (a) without or (b) with dexamethasone (2 /u,M) for 12 h, cells were harvested and detected by TUNEL assay using the In Situ Cell Death Detection Kit (Boehringer Mannheim). Briefly, cells were fixed with 4% paraformaldehyde and permeabilized by proteinase K and incubated with the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT). After washing to remove unbound enzyme conjugated antibody, the horseradish peroxidase retained in the immune complex was visualized by a substrate reaction with diaminobenzidine. The cell nucleus was counterstained with methanol green. Apoptotic eosinophils with nuclear DNA breaks were seen to stain dark brown using a Nikon Eclipse E800 microscope (Nikon Corporation, Tokyo, Japan) in Fig. 8b.

Although the manufacture s instructions directs that the LR reaction should be terminated by addition of the Proteinase K solution as is the case for the BP reaction, this step can be skipped from our experiences. 

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