1. Mechanism of action of cutinase: chemical modification of the catalytic triad characteristic for serine hydrolases
W Köller, P E Kolattukudy Biochemistry. 1982 Jun 22;21(13):3083-90. doi: 10.1021/bi00256a008.
Cutinase from Fusarium solani f. sp. pisi was inhibited by diisopropyl fluorophosphate and phenylboronic acid, indicating the involvement of an active serine residue in enzyme catalysis. Quantitation of the number of phosphorylated serines showed that modification of one residue resulted in complete loss of enzyme activity. One essential histidine residue was modified with diethyl pyrocarbonate. This residue was buried in native cutinase and became accessible to chemical modification only after unfolding of the enzyme by sodium dodecyl sulfate. The modification of carboxyl groups with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide in the absence of sodium dodecyl sulfate did not result in inactivation of the enzyme; however, such modifications in the presence of sodium dodecyl sulfate resulted in complete loss of enzyme activity. The number of residues modified was determined by incorporation of [14C]glycine ethyl ester. Modification of cutinase in the absence of sodium dodecyl sulfate and subsequent unfolding of the enzyme with detergent in the presence of radioactive glycine ester showed that one buried carboxyl group per molecule of cutinase resulted in complete inactivation of the enzyme. Three additional peripheral carboxyl groups were modified in the presence of sodium dodecyl sulfate. Carbethoxylation of the essential histidine and subsequent incubation with the esterase substrate p-nitrophenyl [1-14C]acetate revealed that carbethoxycutinase was about 10(5) times less active than the untreated enzyme. The acyl-enzyme intermediate was stabilized under these conditions and was isolated by gel permeation chromatography. The results of the present chemical modification study indicate that catalysis by cutinase involves the catalytic triad and an acyl-enzyme intermediate, both characteristic for serine proteases.
2. Small interfering RNA for cancer treatment: overcoming hurdles in delivery
Nitin Bharat Charbe, et al. Acta Pharm Sin B. 2020 Nov;10(11):2075-2109. doi: 10.1016/j.apsb.2020.10.005. Epub 2020 Oct 13.
In many ways, cancer cells are different from healthy cells. A lot of tactical nano-based drug delivery systems are based on the difference between cancer and healthy cells. Currently, nanotechnology-based delivery systems are the most promising tool to deliver DNA-based products to cancer cells. This review aims to highlight the latest development in the lipids and polymeric nanocarrier for siRNA delivery to the cancer cells. It also provides the necessary information about siRNA development and its mechanism of action. Overall, this review gives us a clear picture of lipid and polymer-based drug delivery systems, which in the future could form the base to translate the basic siRNA biology into siRNA-based cancer therapies.
3. Characterization of soybean trypsin inhibitor sensitive protease from unfertilized sea urchin eggs
M C Alliegro, H Schuel Biochemistry. 1985 Jul 16;24(15):3926-31. doi: 10.1021/bi00336a018.
A serine protease from sea urchin eggs has been isolated by affinity chromatography on soybean trypsin inhibitor-agarose. Benzamidine hydrochloride was included to minimize autodegradation. We present data on the properties of the protease with respect to molecular weight and its interaction with trypsin inhibitors and substrates. The molecular weight of the enzyme is 47 000 by gel filtration under nonreducing conditions and 35 000 by electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol. The pH optimum and Km with N alpha-benzoyl-L-arginine ethyl ester (BAEE) are 8.0 and 75 microM, respectively. The specific activity is comparable to that of bovine pancreatic trypsin. Proteolytic activity was measured by beta-casein hydrolysis. The caseinolytic activity is completely inhibited by 1 mumol of soybean trypsin inhibitor (SBTI) per micromole of enzyme. BAEE esterase activity is inhibited competitively by SBTI (Ki = 1.6 nM), lima bean trypsin inhibitor (150 nM), chicken ovomucoid (100 nM), and leupeptin (130 nM). Bowman-Birk inhibitor, benzamidine hydrochloride, and antipain are also inhibitors of the purified enzyme. Inhibition by phenylmethanesulfonyl fluoride and N alpha-p-tosyl-L-lysine chloromethyl ketone indicates the presence of serine and histidine residues in the active center, respectively. The chymotrypsin inhibitor L-1-(tosylamido)-2-phenylethyl chloromethyl ketone is ineffective. The protease is susceptible to autodegradation which can result in the appearance of a minor 23-kilodalton component. The egg protease appears to be similar in many respects to trypsins and trypsin-like enzymes isolated from a wide variety of sources, including sea urchin and mammalian sperm.
