Subsites

The esteratic subsite contains the catalytic machinery of the enzyme. The catalytic triad residues - Ser 200, His 440 and Glu 327 (the residue numbering in this section refers to Torpedo californica acetylcholinesterase, TcAChE) - are identical in both enzymes and basically in the same positions. 

The anionic subsite (Ttp 84 and Phe 330) lies between the peripheral and acylation sites, halfway down the gorge and accommodates the positively charged quaternary ammonium of the choline moiety. Ttp 84 orients the charged part of the substrate to the active centre. This subsite is involved in a cross-talk mechanism with the peripheral anionic site (PAS) [3]. 

N-Benzoyl-Lalanine methyl ester is in turn about eight times more reactive than is its D enantiomer). The open-chain compounds may not bind to the enzyme in the same manner, however, as does the locked substrate. The conformation around the amido bond of the open-chain compounds, for example, can be transoid rather than cisoid (81). In addition, if equatorial 24 is considered to be the reactive conformer for both the Dand L enantiomers, and if the alanine methyl group is attracted to the hydrophobic aromatic binding subsite, then structures 34 and 38 would result. The L enantiomer of N-benzoyl-phenylalanine methyl ester 38 in this representation has approximately the same conformation as equatorial L-24. But attraction of the methyl of the D enantiomer to the location occupied by the methyl group of the L enantiomer causes the carbomethoxy group to move from the position it occupies in D-24. 

The locked substrate may bind to the enzyme in a different way from an unrestricted substrate. If the phenyl portion of D-24 binds in the aromatic subsite, then its acylamido group cannot bind at the usual acylamido subsite (82). Cohen justifies this proposal from the fact that some cyclized compounds lacking the acylamido group, e.g., Dmethyl-3,4-dihydroisocoumarin-3-carboxy-late, 39, are good substrates for the enzyme. 

Fig. 7 The influenza virus A sialidase active site showing the five potential inhibitor binding subsites (with S5 containing the hydrophobic pocket formed by reorientation of the Glu276 side-chain), with oseltamivir carboxylate 18 placed in the active site...

The C-6 carboxamide analogues of zanamivir, represented by the general structure 24, provided an avenue to introduce more hydrophobic side-chains onto the dihydropyran scaffold to interact with the hydrophobic regions of subsites S4 and S5 (reviewed in Islam and von Itzstein 2007). The most active tertiary amides (24 = alkyl) showed comparable inhibitory activity to their glycerol side-... 

Importantly, the crystal structure of 34 complexed with N9 sialidase (Fig. 8) indicated differences in the orientation of the guanidino group in subsite S2, and in its interaction with the active site residues, compared to that of zanamivir (Babu et al. 2000). These differences have implications for cross-reactivity of 34 with zanamivir-resistant influenza viruses that have Glul 19 mutations in the sialidase S2 subsite (see Sect. 5.1). 

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