Sialyltransferases reaction catalyzed

Bruner and Horenstein observed a very large inverse -secondary KIE = 0.944 in a reaction catalyzed by a(2 — 6) sialyltransferase (equation 4)." This result demonstrates that, unlike non-enzymatic reactions, there is not necessarily any distance dependence on secondary KIEs in enzymatic reactions enzymes commonly use binding interactions remote from the reaction site to promote catalysis. 

Fig. 20 (a) Inverting reaction catalyzed by a sialyltransferase. (b) Retaining reaction... 

The transfer reaction catalyzed by rat hver a(2 — 6) sialyltransferase using 13 and A-acetyllactosamine as the substrates displayed kinetically significant substrate binding. Even after the observed KIEs for 13 were corrected, they were still too low for any mechanism involving only chemical steps, suggesting that a nonchemical step was kinetically signihcant (Table 5). 

In the third step, the galactosylated invertase (20 mg) was condensed with C-labeled N-acetylneuraminic acid in a reaction catalyzed by sialyltransferase (40 mU) at room temperature (72, 76-77). The... 

Since none of these enzymes have been extensively purified, they are differentiated from one another primarily by the nature of the acceptor and by the linkage synthesized. TTie general reaction catalyzed by the sialyl-transferases is shown in Fig. 9. Although at least four different sialic acids have been described (Gottschalk, 1960a), only two nucleotide sugar derivatives have been isolated, namely, CMP-2V-acetylneuraminic acid and CMP-N-glycolylneuraminic acid it is presently not known whether different enzymes catalyze the transfer of these two different N-acylneu-raminic acids and most of the sialyltransferase literature is limited to the more readily available CMP-N-acetylneuraminic acid. 

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>... 

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. 

Starting from lactose as donor and A -acetylglucose as acceptor substrate the -galactosidase from Bacillus circulans gives exclusively A -acetyl-lactosamine 21 [61], which has been subjected to sialyltransferase-catalyzed conversion to the trisaccharide Neu5Ac-a(2-3)Gal-y8(l-4)GlcA Ac 22 (Scheme 12). By combination of these two enzymatic steps the reaction is pushed toward the product and the problem of low yields related with the galactosidase-mediated reaction could be overcome [62]. 

The total synthesis of sialosides by using the chemoenzymatic approach is as follows [74]. Sialic acid itself can be synthesized from ManNAc, mannose, or their derivatives by sialic acid aldolase enzyme through aldol condensation reaction. If ManNAc is chemically or enzymatically modified at C2, C4—C6 positions, sialic acid has structural modifications at C5, C7-C9 positions, respectively. The sialic acids are subsequently activated by a CMP-siahc acid synthetase to form a CMP-sialic acid, which is the donor used by sialyltransferases. Because CMP-sialic acid is tmstable, the CMP-Neu5Ac synthetase is valuable for the preparative enzymatic synthesis of sialosides. In the last steps, the CMP-sialic acid is transferred to galactose or GalNAc terminated glycosides by sialyltransferases to form structurally defined sialosides. Examples are that Chen and co-workers have recently developed a one-pot multienzyme system for the efficient synthesis of a-sialosides (Table 2) [12,76,79]. In this system, recombinant E. coli K-12 sialic acid aldolase catalyzed the synthesis of sialic acid precursors for... 

Several a3-sialyltransferases have been cloned, and ST3Gal III is commercially available in recombinant form. A particularly interesting report of Gilbert et al. [55] describe a fusion protein consisting of CMP-Neu5Ac synthetase and o2,3-sialyl-transferase from Neisseria meningitidis. This polypeptide was able to catalyze the reactions shown in eqs (2) and (3). 

The regeneration system for CMP-NeuAc is more complicated than that for NDP-sugars (Scheme 7) [24]. An additional phosphorylation step must be incorporated, because CMP, a nucleoside monophosphate, is released after reaction with the sialyltransferase. For recycling purposes, nucleoside monophosphate kinase (NMK EC 2.7.4.4) or myokinase (MK EC 2.7.4.3) is added for the conversion of CMP to CDP. In this reaction, the phosphoryl donor is ATP. Subsequently, both CDP and ADP must be re-phosphorylated to CTP and ATP, respectively. Thus, for regeneration of CMP-NeuAc, an additional kinase and two equivalents of PEP are required. The condensation of NeuAc with CTP is catalyzed by CMP-NeuAc synthetase (EC 2.7.7.43). This system was used for the large-scale synthesis of 6 -sialyl-LacNAc(6 -SLN) from LacNAc catalyzed by a2,6-SiaT (EC 2.7.7.43) in 97% yield. 

In neuroinvasive E. coli K1, synthesis of the a2,8-linked polySia capsule is catalyzed by a CMP-Sia poly-a2,8-sialosyl sialyltransferase (polyST) complex which is postulated to carry out the following reactions ... 

---