Stabilised acyl enzymes

Acyl-a-chymotrypsins may be isolated if the acyl function can be made sufficiently unre-active by structural variation several have been recrystallised at low pH, conditions which stabilise the acyl enzyme. The stabilised acyl enzymes can be characterised by physical methods and the first example of an X-ray crystallographic study of an enzyme intermediate is that of indolylacryloyl-o -chymotrypsin [21]. 

The lysozyme mechanism (Scheme 11.21) has an acylal intermediate with a decay step which is fast compared with its rate of formation, so it is not normally possible to observe the intermediate. The acylal mechanism has recently been established by modifying both enzyme and substrate so that the intermediate can be isolated in this case, the intermediate was stabilised so much that its X-ray crystallographic structure could be determined. Substrate A in Scheme 11.21 was employed [22 ] in which the 2 -fluoro substituent reduces the reactivity at the Cl position so that any acylal enzyme formed from this substrate would have a... 

Applications of low temperature work in structural studies have been described in section 3(b). Application to enzyme action is best exemplified by the pioneering work of Fink and Ahmed [221] and Alber etal. [222] on elastase. JV-Carbobenzoxy-L-alanyl-p-nitrophenol ester was selected for study at — 55°C in a 70% methanol-water mixture. Kinetic studies in the presence of cryoprotectant enabled conditions for formation and stabilisation of the acyl-enzyme intermediate to be established. By monitoring changes in intensity of certain reflections as substrate flowed past the crystal at — 55°C, it was possible to show that the rate of formation of the acyl-enzyme was comparable to that obtained by monitoring p-nitrophenol release spectroscopically. The difference electron density map at 3.5 A resolution showed a peak consistent with the formation of an acyl-enzyme intermediate, but a detailed mechanistic interpretation requires higher resolution data. When the crystal was warmed to — 10°C and the data recollected, the peak in the difference synthesis disappeared, indicating that deacylation had occurred, consistent with the predictions from kinetic studies. 

The enzyme is also subject to allosteric inhibition. When glucose binds at the active site, it stabiles the T-state conformation of the enzyme. The T state is also stabilised by bi- or tricyclic aromatic compounds such as caffeine or flavins, which bind at the entrance to the active site tunnel,and by acylated p-glucopyranosylamine derivatives, which bind similarly to glucose, but more tightly. A third allosteric site, formed at the interface of two subunits and normally an internal pool of water molecules , has recently been discovered in rabbit muscle phosphorylase b " and human liver phosphorylase a. Occupancy of this site freezes the enzyme in the T state and inhibitors with 10 M dissociation constants from the site are being investigated in the treatment of diabetes. 

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