Binding site investigation

Another example of the shape similarity effect on molecular recognition involves the similarity between the structures of the binding sites. Investigation was made for four reaction systems I-IV each consisting of a 1 1 mixture of thiols HS—X and HS—Y... 

Chen, L. H., and Baldwin, T. O. (1989). Random and site-directed mutagenesis of bacterial luciferase investigation of the aldehyde binding site. Biochemistry 28 2684—2689. 

Synthetic nonhydrolyzable analogs of nucleoside triphosphates (Figure 33-13) allow investigators to distinguish the effects of nucleotides due to phosphoryl transfer from effects mediated by occupancy of allosteric nucleotide-binding sites on regulated enzymes. 

With respect to toxins which target specific sites, insight can be obtained from the anomolies that are observed. For example, both puffer fish and tetrodotoxin-containing crabs (96) are insensitive to tetrodotoxin. The investigation of such insensitivities can provide information about membrane channels and their toxin binding sites. 

There has been considerable discussion regarding the mode of action of the sea cucumber and starfish saponins. Both the triterpene and steroidal glycosides inhibit both Na/K ATPase and Ca/Mg ATPase 06) possibly as a result of their aglycone structures. However, their detergent properties cause membrane disruption which will influence the activity of membrane-bound enzymes such as the ATPases. In investigating the actions of saponins on multilamellar liposomes, it was found that cholesterol serves as the binding site for such saponins and that cholesterol-free lip-somes are not lysed by saponins 107). 

The monocationic and dicationic adamantane derivatives have been used to investigate the topography of the channel binding sites of AMPA and NMDA receptors [135]. 

Attention has been drawn to the potential of phosphoric acid anhydrides of nucleoside 5 -carboxylic acids (14) as specific reagents for investigating the binding sites of enzymes. For example, (14 B = adenosine) inactivates adenylosuccinate lyase from E. coli almost completely, but has little effect on rabbit muscle AMP deaminase. The rate of hydrolysis of (14) is considerably faster than that of acetyl phosphate, suggesting intramolecular assistance by the 3 -hydroxyl group or the 3-nitrogen atom. 

The method utilizing ID NMR is simple and eonvenient. Henee the NMR method diseussed here ean be applied to the systematie investigation of the membrane irug inter-aetions, elosely related to the vital function in biomembranes. It is expected that the application can be extended to the lipid-peptide interaction and protein uptake. We are now applying the method to apolipoprotein binding with lipid bilayers and emulsions. Preferential protein binding sites in membranes can be specified by NMR on the molecular level. 

---