A novel photoreactive alpha-amino acid bearing an acidic residue and a cleavable diazirine was developed. To mimic common acidic alpha-amino acids, the residue was designed to be N-acylsulfonamide that possesses an acidic proton and is able to dissociate under the physiological conditions. The inhibition assay of its biotin-tagged derivative with glutamyl endopeptidase from Staphylococcus aureus V8 strain revealed a Ki(app) value of 162 microM, which is slightly higher than the K(m) value of a common substrate. Upon UV irradiation, this derivative specifically photolabeled glutamyl endopeptidase, L-glutamate dehydrogenase, glutamic oxalacetic transaminase, and L-glutamine synthetase, all the enzymes exhibit high affinity toward acidic alpha-amino acids. In addition, N-acylsulfonamide group functioned as a cleavable linker in mild basic solution after a brief N-alkylation. Either the multifunctional nature or the simple structure of this acidic alpha-amino acid surrogate would be useful as versatile photoreactive building block.