Affinity labeling

These labels are not limited to enzymes but may also be designed to react with antibodies or ribozymes although this usage is less common. Although proteins such as hemoglobin do not have an active site, binding pockets can be exploited for their affinity and thus be labeled. Affinity labels can be broken down into three distinct categories based on their reactive groups and mode of delivery. 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. Key to this designation is that the reactivity of the electrophile is not altered by the enzyme and that the noncovalent binding moiety serves to increase the presence and lifetime of the electrophile in the active site (effective molarity). The weakly reactive group may react with functional groups outside of the active site or on other proteins but the selectivity is conferred by the noncovalent binding moiety. Kinetic signatures of this type of inhibitor can be found in saturation because of the covalent reaction (kinact) becomes the rate limiting step at high concentrations of inhibitor. A handful of drugs such as afatinib have gained FDA approval through this approach. The inverse approach of using a weakly nucleophilic inhibitor to attack a protein-bound electrophile has also been studied. This approach has received much less attention due to the lack of protein electrophiles and only those with suitable cofactors can be targeted. Quiescent affinity labels represent a promising approach for inhibiting enzymes using ‘masked’ reactive functionalities that are only uncovered within the active site. This approach differs from mechanism-based inactivators in that the catalysis must be 'off-pathway'. One of the best examples to explain this form of catalysis is in the inactivation dimethylargine dimethylaminohydrolase (DDAH) by 4-halopyridines. At physiological pH, the 4-halo group has near negligible reactivity with thiolates but upon protonation of the nitrogen, the reactivity increases ~4500-fold. This protonation occurs off-pathway by an aspartate residue that is not normally involved in catalysis. Following attack by the active site cysteine and loss of the halide, the enzyme is irreversibly modified. This requirement of catalysis tunes the selectivity of modification. This class is not limited to halopyridines and functional groups including epoxides and peptidyl acyloxymethyl ketones have been used. The kinetic signature of this class resembles that of classical affinity labels.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.