Chymotrypsin protease inhibitor

Phenylmethanesulfonyl fluoride (PMSF) [329-98-6] M 174.2, m 90-91 , 92-93 . Purified by recrystn from ""CgHe, pet ether or CHCl3-pet ether. [Davies and Dick J Chem Soc 483 1932 cf Tullock and Coffman J Org Chem 23 2016 I 960.] It is a general protease inhibitor (specific for trypsin and chymotrypsin) and is a good substitute for diisopropylphosphoro floridate [Fahrney and Gould 7 Am Chem Soc 85 997 1963]. 

Coumarincarboxylate derivatives are versatile, efficient, low molecular weight, nonpeptidic protease inhibitors. Both esters and amides behave as time-dependent inhibitors of a-chymotrypsin but the esters are clearly more efficient than the corresponding amides. The criteria for a suicide mechanism are met. The presence of a latent alkylating function at the 6-position (chloromethyl group) is required to produce to inactivation by a suicide mechanism (Scheme 11.3, pathway a). Aryl esters, in particular the meta-substituted phenyl esters are the best inhibitors. Thus, m-chlorophenyl 6-(chloromethyl)-2-oxo-27/-l-benzopyran-3-carboxylate is one of the well-known inactivator of a-chymotrypsin (kJK, = 76(),000M s 1 at pH 7.5 and 25 °C, Table 11.1). 

A large number of potential reversible protease inhibitors exist (Laskowski Kato, 1980). Protein protease inhibitors like Strepromyces Subtilisin Inhibitor (SSI) (Hiromi et al, 1985) and Chymotrypsin Inhibitor (CI-2) (Jonassen, 1980 and McPhalen James, 1988) are known to be very strong inhibitors with inhibition constants at or below 10"10 M. 

Serine protease inhibitor that inhibits trypsin, chymotrypsin, kallikrein, and plasmin Binding is reversible, with most aprotinin-protease complexes dissociating at pH>10or <3 Peptidase inhibitor... 

Cyanobacteria -Microcystis aeruginosa Micropeptins 478-A and -B -peptide-based plasmin inhibitors Cyanopeptolins A-D -protease inhibitors Cyanopeptolin 963A-chymotrypsin inhibitor 120 6, 262 263... 

Potl inhibitors differ from other protease inhibitors, and from all other defense peptides mentioned thus far, in their relative lack of disulfide bonds. This means that the loop with the reactive site is not fixed, as it is in the Bowman-Birk inhibitors, yet they still form a stable fold, as shown in Figure 11. An interesting feature of some Potl inhibitors is their tendency to form stable, noncovalently bound oligomers. This has, for example, been shown for chymotrypsin inhibitor I from tomato. This peptide has a monomer weight of 8300 Da under dissociating sodium dodecyl sulfate (SDS) gel conditions. Gel filtration and ultracentrifugal analysis revealed a... 

Potll inhibitors inhibit several proteases. For example, serine protease inhibitors isolated from Capsicum annum seeds named PSI-1.1 and PST1.2 are strong inhibitors of trypsin and only one order of magnitude less active on chymotrypsin. PSTl.l also has considerable activity against thrombin, whereas factor Xa is inhibited to a lesser extent by PSI-1.1 and PSTl.2. ... 

Because PMSE fails to inactivate acetylcholinesterase, this reagent is much less toxic than diisopropylfluoro-phosphate, and is also recommended as an alternative to the neurotoxic fluorophosphates and fluorophospho-nates. PMSE is freshly prepared as a 1-3 mM solution in water (higher concentrations will precipitate spontaneously). A better procedure is to first prepare a 20 mM PMSE solution in 2-propanol or dioxane this solution can then be added to the biological fluid with vortex mixing to achieve a 1-3 mM final concentration as a homogeneous solution. One should confirm that the alcohol or dioxane has little or no undesirable effect on enzymes or proteins of interest. See Chymotrypsin Protease Inhibitor Cocktails ... 

Frenkel, K., Chrzan, K., Ryan, C. A., Wiesner, R., Troll, W. (1987). Chymotrypsin-speeifie protease inhibitors deerease H2O2 formation by aetivated human polymorphonuelear leukoeytes. Carcinogenesis, 8, 1207-1212. 

Sequences have been determined for plasminogen and bovine Factor XII, and they are not homologous with the other serine proteases. The amino-terminal sequence of Factor XII is homologous, however, with the active site of several naturally occurring protease inhibitors (11). Numbering corresponds to chymotrypsin with active serine at 195. 

Protease nexin 2 is identical to the secreted form of the amyloid precursor protein containing the Kunitz-type serine protease inhibitor domain (128,129), Protease nexin 2 circulates in blood stored as a platelet a-granule protein, which is secreted upon platelet activation (127). Protease nexin 2 inhibits trypsin- and chymotrypsin-like serine proteases and is also a potent inhibitor of factor Xla (126,127,128). Its location in platelets and its ability to inhibit factor Xla suggests a role in regulating blood coagulation for protease nexin 2. 

The Bowman-Birk type protease inhibitors represent a class of low molecular weight, cysteine-rich proteins found in legume seeds (.10). The major Bowman-Birk inhibitor in soybean seeds is a double-headed protein capable of blocking the activity of both trypsin and chymotrypsin. This protein represents approximately 4% of the total protein in soybean seeds (1J ). In contrast to the soybean trypsin inhibitor (Kunitz), the "double-headed inhibitor (referred to as BB) is typical of protease inhibitors present in a large number of legume seeds for example, peanuts (12) chick peas (33)5 kidney beans (3JO adzuki beans (33) lima beans (16). 

There is evidence that protein structures are also responsible for cell cohesion in nonpalmo-plantar stratum corneum. When punch biopsies of normal human gluteal skin were incubated in a buffer containing a mixture of the zwitterionic surfactant /V,/V,-dimethyldodecylamine and the anionic surfactant sodium dodecyl sulfate,11 there was dissociation of cells in the stratum corneum but not in the rest of the epidermis. The cell dissociation took place only in the presence of EDTA and was inhibited by the serine protease inhibitor aprotinin.12 Suzuki et al.13,14 presented evidence that spontaneous cell dissociation in nonpalmo-plantar stratum corneum could be inhibited by a combination of inhibitors of trypsin-like and chymotrypsin-like enzymes. Thus, nonpalmo-plantar stratum corneum contains endogenous proteases that mediate cell dissociation. 

Peptides that display a terminally located aldehyde function in their structure constitute another group of modified peptide enzyme inhibitors. The sequence benzyloxycarbonyl-Pro-Phe-CHO fulfils the known primary and secondary specificity requirements of chymotrypsin and has been found to be a potent reversible inhibitor of this proteolytic enzyme (Walker et al. 1993). Further, protease inhibitors comprising terminally located aldehyde function are antipain, leupeptin, chymostatin, and elastatinal. In addition, also phos-phoramidon, bestatin, puromycin, and amastatin represent modified peptides which can reversibly inhibit enzymes. 

Amongst the subnanomolar chymotrypsin inhibitors, modelling of one of the best variants implied a novel inhibitory mechanism for protein serine protease inhibitors, in which two amino acid side chains (arginine and aspartic acid) intrude into the proximity of the catalytic triad of the protease rather than binding in the substrate-binding pockets (see Fig. 4). 

This survival strategy may be more complex, however, as a number of studies 376, 377, 515, 516, 591-593, 744, 832, 891) have indicated that pseudophyllidean and cyclophyllidean cestodes produce their own protease inhibitors which can inactivate trypsin and chymotrypsin in vitro. Such protease inhibitors are also produced by nematodes and they appear to be proteins or protein-carbohydrate complexes 592). Precise chemical characterisation of these protease inhibitors has not yet been achieved in cestodes, although the active molecule from the strobilocercus of T. taeniaeformis has been shown to be a polypeptide taeniaestatin) with an Mt of 19 500 under reducing conditions 832). The effectiveness of this molecule at inhibiting chymotrypsin in vitro is illustrated in Fig. 4.1. The mechanism whereby this and... 

Angiotensin I converting enzyme Acquired immunodeficiency syndrome Aspartic protease inhibitor Bowman Birk protease inhibitor protein Chymotrypsin Cysteine protease inhibitor Endothelin-converting enzyme Elastase... 

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