Enzyme complex intermediates, glycosyl

Glycosyl—Enzyme Complex Intermediates in Biosynthesis of Complex Saccharides. The synthesis of nucleoside diphosphate sugars involves the transfer of a nucleotidyl group from a nucleoside triphosphate to a sugar 1-phosphate with the simultaneous release of pyrophosphate according to the following general reaction (11) ... 

Group (c), a-D-mannosidase from jack beans and from almonds, and a-D-galactosidase from coffee beans, showed no inactivation. The results with these enzymes can possibly be explained by the formation of a (weak) non-covalent complex in which glycosylation is too slow to cause inactivation within the time period of measurements, or, less likely, rapid hydrolysis of the glycosyl-enzyme intermediate. 

For the study of polyprenyl glycosyl phosphates as intermediates in the synthesis of complex glycans, several techniques have been developed, and these have been described elsewhere in detail.18 20 Two important features should be emphasized. First, the very small amounts of polyprenyl phosphates that are present in most tissues for this reason, the use of radioactive techniques for the detection of products is obligatory. Second, on account of the hydrophobic nature of these compounds, and as the enzymes involved in the reactions are membrane-bound, the use of detergents and organic solvents becomes necessary. 

Kis in this case is not, however, the dissociation constant of the second molecule of substrate from the ES2 complex, but Kd[(k+2 + k+3)lk+2], where is the dissociation constant of the glycosyl-enzyme intermediate. 

GH 2. The tetrameric lacZ (3-galactosidase of Escherichia coli has been intensively investigated for over 40 years, initially because of its relationship to Monod s classic work on enzyme induction. A very large set of data accumulated by Wallenfels and Weil unfortunately had to be discarded because it was obtained in inhibitory Tris buffers.The first glycosyl-glycosidase intermediate to become kinetically accessible was with this en-zyme the 2-fluoro-2-deoxy-a-D-galactopyranosyl enzyme, the parent enzyme and ES complexes have since been crystallised and its structure determined. 

The exact sugar conformation in the ES complex of GH 33 enzymes seems to vary from substrate to substrate. Thus, complexes of 4-methylumbelliferyl N-acetyl-a-neuraminide in complex with the (inactive) acid-base mutant of the T. cruzi tf(2 .s-sialidase reveal a conformation, whereas the complex with sialyllactose has a 82 5 ring these are next to each other on the pseudoro-tational itinerary (Figure 2.6b). A possible conformational trajectory for the pyranose ring in these enzymes is 82 5 (ES complex) (probably most stable conformation of the sialosyl cation, with NElAc and OH pseudoequa-torial) Cs (glycosyl-enzyme intermediate). This would result in most of the motion of the substrate relative to enzyme in the course of catalysis being in the carbon which is being substituted, in the normal way. 

There is no direct evidence on the form of the glycosyl-enzyme intermediate. Nonetheless, stabilization of a noncovalent oxocarbonium for the period between product fructose release and acceptor binding may not be realistic for slow reactions, given the extremely short lifetime of a glycosyl oxocarbonium ion (9). Levansucrase and particularly GTase-S are quite slow enzymes, with sucrose hydrolysis Heat of 48 sec 122) and 9.1 see" 213), respectively. Thus, the carbonium ion may well collapse to a more stable covalent complex or develop an equilibrium between the two forms. Nucleophilic catalysis is consistent with... 

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