In order to determine which amino acids are involved in substrate binding, castor bean seeds acid phosphatase was treated with amino acid-modifying reagents, such as phenylglyoxal (PGO), iodoacetic acid (IAA), N-bromosuccinimide (NBS), N-acetylimidazole (NAI), diethylpyrocarbonate (DEPC), N,N'-dicyclohexylcarbodiimide (DCCD) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), specific for arginine, cysteine, tryptophan, tyrosine, histidine, and aspartic and glutamic acids, respectively. Enzyme activity was determined using p-nitrophenylphosphate (pNPP) as substrate. Enzyme inhibition was observed with IAA, NBS, EDC and DCCD. In the presence of the reaction products, p-nitrophenol (pNP) and inorganic phosphate (Pi), which are competitive inhibitors, the enzyme was protected from inactivation by IAA, indicating involvement of the enzyme active site. The inhibition by IAA was time- and concentration-dependent, with an apparent bimolecular rate constant of 48 x 10(-4) M-1 s(-1),and two molecules of IAA bound per active site. Our results suggest that sulfhydryl groups are essential for enzyme catalysis, and are located in or near the substrate-binding domain. Other amino acids such as tryptophan, aspartic and glutamic acids, were also important for the enzyme activity, but were probably located outside of the active site. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.1644629633