Inhibitors, enzyme cysteine proteinase

In this laboratory, we also include the metal ion chelators EDTA (ethylene diamine tetraacetic acid binds, e.g., Mg2 1 -ions) and EGTA (ethylene glycol-bis(2-aminoethyl)-Al,iV,iV/,iV/,-tetraacetic acid binds, e.g., Ca2+-ions) in our lysis buffers. These agents help prevent phosphatase action (by the metal ion-dependent phosphatase PP2C, which is not inhibited by microcystin-LR), metal (Ca2+) dependent proteinases, and protein kinases, which require divalent cations such as Mg2 1 (and, in some cases, also Ca2+). We also use a mix of proteinase inhibitors that inhibit a broad range of proteolytic enzymes, including serine and cysteine proteinases. 

Crystallographic studies of native cysteine proteinases and enzyme-inhibitor complexes have been used to interpret much or the kinetic data for cysteine protemsse-caUlyzed hydrolysis of amide bonds. Analysis of the crystal structures of papain [16]. caricain [38], actinidain [56], etc. shows that these structures are closely related. The active site of all these cysteine proteinases contains the Cys-25 sulfhydryl group in close proximity to the His-159 imidazole ring nitrogens, where the latter can abstract the sulfhydryl proton to facilitate attack on the substrate amide carbonyl group [17]. 

A potent cysteine proteinase inhibitor PCPl 8.3 was isolated from potato tubers. The inhibitor has a broad inhibitor spectrum, including the bromelain enzymes, which am not inhibited by cystatin. PCPI shows a Ki value of 190 nM for stem bromelain, 33 nM for firuit bromelain, 0.06 nM for ananain, and 3.3 nM for papain [78]. ft is proposed (hat the differences of inhibitory spectrum between PCPl and the cystatins may be those of distinct supeifamilies of cysteine proteinase inhibitors. 

Al. Abrahamson, M., Barrett, A. J., Salvesen, G., and Grubb, A., Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids. J. Biol. Chem. 261(24), 11282—11289 (1986). 

H3. Hall, A., Abrahamson, M., Grubb, A., Trojnar, J., Kania, P., et al., Cystatin C based peptidyl diazomethanes as cysteine proteinase inhibitors Influence of the peptidyl chain length. J. Enzyme Inhib. 6(2), 113-123 (1992). 

Part Three reviews representative multienzyme compositions, such as pancreatin, as well as promising recent developments with glucocerebrosidase, deoxyribonuclease and protein inhibitors of elastase. This part integrates biochemical, experimental, and clinical data of therapeutic enzymes, such as asparaginase, bromelain, hyaluronidase, and cysteine proteinases, about which... 

Mouse and chick bone explants have been shown to produce a latent collagenase.33,34 jn addition, the mouse bone explants produce a latent neutral proteinase. Limited proteolysis of these molecules gives rise to proteoglycan and collagen degrading action. Latency may be due to the presence of an enzyme inhibitor complex. The chick latent collagenase has an apparent MW of 54,000, while the active form has an MW of 43,000. 3 Trypsin activation of both latent mouse enzymes decreases their MW s from 60-70,000 to about 40,000. The mouse neutral protease is inhibited by EDTA, cysteine, and serum.34... 

A purification and some properties of proteinase A from yeast are described. A specific macromolecular inhibitor of proteinase A from yeast cytosol has been isolated and shown to be a protein (molecular weight 7,700) consisting of a majority of polar amino acids. Proline, arginine, cysteine and tryptophan were not detected in the inhibitor. Possible biological functions of proteinase A and the proteinase A-inhibitor (and of other yeast proteinases and their inhibitors) in the following processes are discussed general protein turnover, catabolite inactivation of enzymes, enzyme degradation at starvation and at transition to spore formation, and activation of pre-enzymes and precursor proteins by limited proteolysis. 

The L-aminopeptidase is sensitive to various classes of proteinase inhibitors. Strong inhibition of the enzyme is observed by treatment with the thiol reagents / -chloromercu-ribenzoate (pCMB) and iodoacetamide. The inhibition by pCMB can be reversed by subsequent treatment with dithiothreitol. In addition, the enzyme is inhibited by the metalchelating compounds EDTA and o-phenanthroline and the serine protease inhibitors phenylmethylsulfonyl fluoride and diisopropylfluorophosphate. These phenomena point to an essential serine or cysteine residue in the active site furthermore, divalent cations seem to be involved in the catalytic mechanism and/or are important for the stability of the enzyme. 

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