Essential sulfhydryl groups in the active site of castor bean ( Ricinus communis) seed acid phosphatase

Abstract

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 48×10 −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.