Molecular docking experiments with putative substrate of enzyme could confirm purported enzyme active site

Active site of an enzyme provides a tailored environment for enhancing the effectiveness of substrate to product or intermediate conversion. Armed with amino acid residues, a first contact shell and catalytic moieties, the active site is the first target of enzymologists when they examine a recently published crystal structure of an enzyme. But, how do we identify an enzyme active site? One approach is through molecular docking experiments with putative substrate of the enzyme. Specifically, a 3D molecular model of the substrate is constructed and used as ligand for probing different aspects of the crystal structure of the enzyme. Grooves and pockets that just fit or accommodate the substrate would be candidates as active site of the enzyme. However, some enzymes have a broad substrate scope that allows it to catalyse reaction of a range of different substrates of different molecular size. Hence, size alone may not provide sufficient constraints for identifying the correct active site from amongst a couple of candidates. An additional criterion that could help narrow the search for the correct active site is the orientation in which the substrate binds to specific moieties or catalytic centre of the active site. If different substrates of different size bind the putative active site in a particular orientation that brings a functional group of the substrate into close contact with the catalytic centre, the putative active site may rank highly as the correct active site of the enzyme. Hence, advancement in molecular modelling and simulation tools have afforded molecular docking as a feasible approach for identifying and even confirming putative enzyme active site with respect to a class of substrates. In this case, size of binding pocket serves as an initial criterion for narrowing the search of putative active site for a substrate, but, more refined analysis would need the support of binding orientation of substrate in binding pocket to nail down the definitive active site of an enzyme.