Substrate activity screening

This is a fragment screening strategy in which substrates for a particular target protein are identified and then optimized rapidly. Eor example, substrates for the cysteinyl protease cathepsin S bearing a fluorogeiuc group were optimized for 

In some cases, it is possible to observe ligand-protein complexes of fragment libraries by electrospray ionization mass spectrometry. This method was used to identify novel hits that functionally inhibit bacterial protein synthesis by binding to a subdomain of the 23S rRNA subunit. 

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A. Binding energy as the sum of analysis A. Substrate activity screening... 

Wood, W. J. L., Patterson, A. W, Tsirruoka, H., Jain, R. K., Ellman, J. A. Substrate activity screening a fragment-based method for the rapid identification of nonpeptidic protease inhibitors. J. Am. Chem. Soc. 2005,127, 15521-15527. 

Hornsby, M., Lesley, S., Spraggon, G., and Ellman, J.A. (2006) Identification of selective, nonpeptidic nitrile inhibitors of cathepsin S using the substrate activity screening method. Journal of Medicinal Chemistry, 49 (21), 6298-6307. 

Patterson, A.W, Wood, W.J.L., and Ellman, J.A. (2007) Substrate activity screening (SAS) a general procedure for the preparation and screening of a fragment-based non-peptidic protease substrate library for inhibitor discovery. Nature Protocols, 2 (2), 424—433. 

In using this substrate to screen activity in a range of commercial pectinase preparations, it was found that for all preparations, except two, the same ratio of activity on this substrate and linear arabinan (from pear juice) was obtained as was obtained for pure em/o-arabinanase. One of these preparations was Novo Ultra SP. This preparation hydrolysed CM-Debranched Arabinan at 1.5 times the rate of pear-juice linear arabinan. This additional... 

It is emphasized that in the case of kinetic resolution, the MS measurements must be performed in the appropriate time window (near 50% conversion). If this is difficult to achieve due to different amounts or activities of the mutants being screened in the wells of microtiter plates, the system needs to be adapted in terms of time resolution. This means that samples for MS evaluation need to be taken as a function of time. Finally, it is useful to delineate the possibility of multi-substrate ee screening using the MS-based assay, which allows for enzyme fingerprinting with respect to the enantioselectivity of several substrates simultaneously. 

Substrate cocktail [61-63] 10 CYP-selective substrates/ reactions to measure major CYP enzyme activities Quick activity screens Possible interactions between substrates... 

One of the reactions catalyzed by esterases and lipases is the reversible hydrolysis of esters (Figure 1 reaction 2). These enzymes also catalyze transesterifications and the asymmetrization of meso -substrates (Section 13.2.3.1.1). Many esterases and lipases are commercially available, making them easy to use for screening desired biotransformations without the need for culture collections and/or fermentation capabilities. As more and more research has been conducted with these enzymes, a less empirical approach is being taken due to the different substrate profiles amassed for various enzymes. These profiles have been used to construct active site models for such enzymes as pig liver esterase (PLE) (EC 3.1.1.1) and the microbial lipases (EC 3.1.1.3) Pseudomonas cepacia lipase (PCL), formerly P.fluorescens lipase, Candida rugosa lipase (CRL), formerly C. cylindracea lipase, lipase SAM-2 from Pseudomonas sp., and Rhizopus oryzae lipase (ROL) [108-116]. In addition, x-ray crystal structure information on PCL and CRL has been most helpful in predicting substrate activities and isomer preferences [117-119]. 

Tambor JH, Ren H, Ushinsky S, Zheng Y, Riemens A, St-Francois C, Tsang A, Powlowski J, Storms R. (2012). Recombinant expression, activity screening and functional characterization identifies three novel endo-l,4-P-glucanases that efficiently hydrolyze cellulosic substrates. Appl Microbiol Biotechnol, 93, 203-214. 

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