Sialyltransferase reaction

The stimulatory effect of nonionic detergents on the sialyltransferase reactions may reflect an interaction of the hydrophobic environments of the active sites with the detergents, possibly by the insertion of the latter into the lipid bilayer surrounding the enzymes or by the formation of detergent-enzyme complexes, thus inducing more active enzyme conformations (51,52,53). The effect of nonionic detergents may be similar to the previously reported effects of phosphatidyl ethanolamine (34), CDP-choline and lysolecithin (54,55), phospho-diglycerol and cardiolipid (56). 

Inhibition of enzyme activity may be due to substrate, product or other effector molecules, and the concentrations of these components in the cell may lead to regulation of the pathway. Competitive product inhibition has been ascribed a significant role in sialyltransferase reactions (Bernacki 1975, Klohs et al. 1979, Eppler et al. 1980 b) with CMP yielding apparent Kj values of 50 (xM (Klohs et al. 1979). This inhibition may be a self-regulatory effect in situ in view of the high nucleotide phosphatase activity of Golgi apparatus. A non-competitive inhibition of sialyltransferase activity was also observed with other nucleotides (Bernacki et al. 1978, Klohs et al. 1979). 

Hematoside in Hamster Cells. Hakomori and Murakami (1968) reported that the levels of hematoside, the only ganglioside in BHK, was reduced in spontaneous and Py-transformed BHK cells. Similar results were observed with BHK cells transformed by two strains of Rous sarcoma virus (RSV) (Hakomori et aL, 1968). The reduced hematoside content in at least the Py-BHK cells was attributed to decreased activity of lactosylceramide CMP-NANA sialyltransferase, reaction 1 in Figure... 

Example 5 Hayakawa and Noyori group in their studies on new activators for phosphoroamidite coupling reactions have applied the most effective member of the group of acid/azole complexes AT-(phenyl)imidazolium tri-flate (N-PhIMT) in the efficient synthesis of biologically important compounds [20j]. A noteworthy example is synthesis of cytidine-5 -monophos-pho-AT-acetylneuraminic acid. This compound is a source of sialic acid in the sialyltransferase-catalysed biosynthesis of sialyl oligosaccharides [25]. 

O-SIALOG LYCOPROTEASE SIALYLTRANSFERASE SIDE REACTIONS SIGMA (<7)... 

The regeneration system for CMP-NeuAc can be employed both for a2,3-sialyltransferase-catalyzed reactions and for reactions mediated by a2,6-sialyltransferase. The system starts with NeuAc, the glycosyl acceptor, PEP, and catalytic amounts of ATP and CMP. CMP is converted to CDP by nucleoside monophosphate kinase (EC 2.7.4.4 NMK) in the presence of ATP, which is regenerated from the by-product ADP, catalyzed by PK in the presence ol PEP, then to CTP with PEP by PK. The CTP thus formed reacts with NeuAc, catalyzed b>... 

CMP-NeuAc synthetase (EC 2.7.7.43) to produce CMP-NeuAc. The by-product pyrophosphate (PPi) is hydrolyzed to phosphate (Pi) by inorganic pyrophosphatase (PPase). Sialyla-tion is accomplished with a2,3-sialyltransferase (< 2,3NeuAcT) or a2,6-sialyltransferase (a2,3NeuAcT), respectively. The released CMP is again converted to CDP, to CTP, and finally to CMP-NeuAc. The UDP-Gal and CMP-NeuAc regeneration schemes have been combined in a one-pot reaction and applied to the synthesis of sialyl Lewis X. 

It is interesting that all four reactions studied gave the same apparent Km values for CMP-NeuNAc. This observation suggests that the CMP-NeuNAc binding sites of the different species of sialyltransferases are similar in structure and affinity properties, although the binding sites for the acceptors and the catalytic sites may be very different, providing a basis for substrate specificities. 

Chemical synthesis of sialosides is considered one of the most difficult glycosylation reactions because of a hindered tertiary anomeric carbon and the lack of a participating auxiliary functionality in the carbon next to the anomeric carbon in sialic acids (55, 56). Sialyltransferase-catalyzed glycosylation is believed to be the most efficient approach for the production of sialic acid-containing structures. 

A corresponding approach has been employed for a chemoenzymatic synthesis of Neu5Aca2-6Galy3l-4GlcNAc using -galactosidase from Bacillus circulans and a2-6-sialyltransferase from rat liver [79]. In this reaction, less complicated problems had to be solved than in the above case in which the pH optima of all enzymes were in a wider range. 

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