The hallmark of an affinity label is the use of a targeting moiety to specifically and reversibly deliver a weakly reactive group to the enzyme that irreversibly binds to an amino acid residue.
The targeting portion of the label often resembles the enzyme's natural substrate so that a similar mode of noncovalent binding is used prior to the covalent linkage.
[3] This category encompasses the simplest approach of coupling an electrophile with low intrinsic reactivity to a noncovalent binding moiety which frequently mimics the natural substrate.
[1][3] Quiescent affinity labels represent a promising approach for inhibiting enzymes using ‘masked’ reactive functionalities that are only uncovered within the active site.
This term has been previously used to describe affinity labels that contain weakly reactive groups but recent literature has commenced on the requirement of off-pathway catalysis.
While it is clear that X-ray crystallography will provide more detailed 3-D information about the active site, only a static picture is returned and difficulties can be encountered with co-crystallization of the substrate or mimics due to enzymatic turnover.
[1] Another notable example of using affinity labeling to determine the active site of an enzyme is the work carried out by Grachev et al. which resulted in characterization of the β-subunit of the core RNA polymerase as the sub-unit responsible for phosphodiester-bond formation in the process of prokaryotic transcription.