Active site specificity

A closer examination of these essential residues, including the catalytic triad, reveals that they are all part of the same two loop regions in the two domains (Figure 11.10). The domains are oriented so that the ends of the two barrels that contain the Greek key crossover connection (described in Chapter 5) between p strands 3 and 4 face each other along the active site. The essential residues in the active site are in these two crossover connections and in the adjacent hairpin loops between p strands 5 and 6. Most of these essential residues are conserved between different members of the chymotrypsin superfamily. They are, of course, surrounded by other parts of the polypeptide chains, which provide minor modifications of the active site, specific for each particular serine proteinase. 

Another property relevant to the current discussion is the distribution of water in the active site. Specifically, we characterize the population of various water wires connecting the zinc-bound water/hydroxide and His 64 found in the SCC-DFTB/MM simulations. These wires were identified following a definition of hydrogen-bond in terms of both distance (O—O < 3.5 A) and angle (O—H—O > 140°) and care... 

Recently, imino sugars have found application as active site specific chaperones (ASSC) for the treatment of lysosomial storage disorders.65 An ASSC is a small molecule that bind the catalytic domain of an enzyme inducing the regeneration of the active conformation of misfolded proteins. 

J.-Q. Fan, Imino sugars as active site specific chaperones for the treatment of lysosomial storage disorders, in Imino sugars from Synthesis to Terapeutic Applications, ed. P. Compain and O. Martin, Wiley, 2007, pp. 225-247. 

Treatment of 9-(/ -D-ribofuranosyluronic acid)adenine with diphenylphosphoro-chloridate and orthophosphate or tripolyphosphate yields (62) and (63), which, although unstable, inhibit rabbit AMP aminohydrolase and pyruvate kinase, respectively, with behaviour characteristic of active-site-specific reagents.98 Adenylate kinases from several sources are inactivated by iV6-[2- and 4-fluorobenzoyl]-adenosine-5 -triphosphates, with kinetics characteristic of active-site labelling, although these compounds were without effect on yeast hexokinase and rabbit pyruvate kinase.99... 

The two proteinase Inhibitors that accumulate In leaves of wounded tomato leaves have been Isolated and characterized. Inhibitor I has a molecular weight of 41,000 and Is composed of subunits with molecular weights of about 8100 (10). It Is, therefore, a pentamer In Its native state. Each subunit possesses an active site specific for chymotrypsln, and the apparent for the Inhibition of chymotrypsln Is about 10 M (10). Inhibitor II has a molecular weight of about 23,000, Is composed of two subunits, and strongly Inhibits both trypsin and chymotrypsln with Kj values of about 10 and 10 M respectively... 

The study of both heterogeneous catalysts and enzymes is dominated by the concept of the active site. Specifically, in enzymes the active site is known to represent only a small portion of the large protein molecule that is the enzyme [6], The active site may lie at or near the surface, but it may also be buried in an active site groove or crevice that limits access of all but the desired substrate. Clearly, the total surface area of the protein is significantly larger than that of the active site. 

Feil, R., Brocard, J., Mascrez, B., LeMeur, M., Metzger, D., and Chambon, P. (1996) ligand-activated site-specific recombination in mice. Proc. Natl. Acad. Sci. USA 93, 10887-10890. 

Search the Enzyme Structure Database for y-chymotrypsin active site (by the aid of the active-site-modified enzyme or active-site-specific inhibitor-enzyme complex) to identify and depict (save pdb file) the catalytic triad of y-chymotrypsin. 

Proteolytic Enzymes and Their Active-Site-Specific Inhibitors Role in the Treatment of Disease... 

Apart from lAPs, there are several nonmammalian regulators of caspases, which are active-site specific inhibitors (Callus and Vaux, 2007). One example is a serpin from the cowpox virus, cytokine response modifier A (crmA). CrmA forms a covalent complex with the initiator caspase-1 and -8 resulting in irreversible inhibition of these caspases. It also inhibits caspase-6 but less efficiently (Dobo et al., 2006). The baculoviral protein p35 is a broad spectrum caspase inhibitor that irreversibly inactivates caspases (Bump et al., 1995 Fisher et al., 1999). 

Twu, J.S., Wold, F. (1973). Butyl isocyanate and active site specific reagent for yeast alcohol dehydrogenase. Biochemistry 12 381-6. 

Acid proteases are inactivated by active-site specific reagents, diazoacetylnorleucine ethyl ester and other diazo compounds, and epoxy (p-nitrophenoxy)propane. Covalently labelled aspartic acid peptides have been isolated from pepsin, chymosin (= rennin), and penicillopepsin. The peptides labelled with the diazo compounds have similar sequences and differ from the epoxy (p-nitrophenoxy)pro-pane labelled peptides. These results indicate two aspartic acids at the active site and suggest homology between the enzymes. The latter is confirmed by a comparison of the sequence data. Studies of the action of porcine pepsin and penicillopepsin on some dipeptides with free N-terminal groups show transpeptidation involving a covalent acyl intermediate. It is proposed that there are differences in the mechanism of action of pepsin which are determined by the nature of the substrate. 

Martensen TM, Mansour TE. Studies on heart phosphofructoki-nase. Use of fructose 6-sulfate as an alternative substrate to study the mechanism of action and active site specificity. J. Biol. Chem. 1976 251 3664-3670. 

Captopril angiotensin converting enzyme competitive, active site specific ACE- 23 nM (IC50) aminopeptidaseP-110 ixM (45), (46)... 

Chang HH, Asano N, Ishii S, Ichikawa Y, Fan JQ. Hydrophihc 44. iminosugar active-site-specific chaperones increase residual glucocerebrosidase activity in fibroblasts from Gaucher patients. FEBS... 

Fan JQ, Ishii S. Active-site-specific chaperone therapy for Fabry disease. Yin and Yang of enzyme inhibitors. FEES J. 2007 274 4962-4971. 

Structural Insights, Chymotrypsin A Serine Protease. Work with interactive molecular models to learn more about the structural bases of active site specificity and reactivity, and some of the ways in which active site residues can be identified. 

M Lackmann, et al. Radioimmunoassay for the detection of active site specific thrombin inhibitors in biological fluids—Heparin affects the binding of hirudin to alpha-thrombin. Thromb Res 63 609, 1991. 

The perceived solution to this problem was to insert a second catalytic center into the active site specifically to carry out the dephosphorylation step of the reaction. Applying this rationale, the human form of BChE has been mutated (Figure 7.3) to... 

P. Chambon, Ligand-activated site-specific recombination in mice,... 

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