Although some of the examples of channelling cited by Ov/tdi (1991) may be controversial, tryptophan synthase is an example where the data support of substrate channelling seem unequivocal (for recent reviews see Miles, 1991 and Miles et al., in press). Direct evidence comes from the 3-D structure from tryptophan synthase from Salmonella typhimurium and shows a 25 ,~ long hydrophobic tunnel (Hyde et al., 1988). Hence the review by Ovfidi needs correction, in her table 2 the X-ray crystallographic structure is not from Escherichia coli. In fact attempts to crystallize enzyme from E. coli have not been very successful.
The reason for the required substrate channelling of indole appears to be obvious. The tunnel can accommodate up to four molecules of indole. Indole is very hydrophobic, and if released from the enzyme's active site, it would dissolve in the cell membranes. Thus, indole is channelled to preclude the loss of an important metabolite and possible toxic side effects.
Kinetics of substrate channelling have been recently monitored directly by chemical quench flow and stop-flow methods. Anderson et al. (199 I) have shown that efficient channelling of indole through channelling is due to the following reasons: (a) the rate of diffusion of indole through the channel is very fast; (b) the reaction of indole to form tryptophan at the fl site is fast and largely irreversible; and (c), the reaction of serine at the fl site modulates the formation of indole at the a site or in the tunnel such that indole is not produced until serine has reacted with pyridoxal phosphate to form highly reactive aminoacrylate. This intersubunit communication keeps the a and fl reactions in phase so that indole does not accumulate at the a active site.
A mutant form of the a2f12 complex with an engineered restriction in the tunnel (fl C170F) accumulates indole (Miles et al., in press and Anderson, Yang and Miles, in preparation).
Rapid kinetic studies also substantiate the channelling of indole in the aft reaction. Free indole from solution may pass via the a site and the interconnecting tunnel to the fl site. This hypothesis is favoured by rapid-scanning stopped-flow investigations that show that a ligand that binds to the a site, a-glycerol-3-phosphate, decreased rate but not the yields of the reaction of indole at the fl site (Dunn et al., 1990). The decreased rate may result from ligand induced conformational change that restricts the access of indole to the a site or to the tunnel.