The effect of amino acid modifying reagents on the activity of a (1→3)-β-glucan synthase from Italian ryegrass (Lolium multiflorum) endosperm

Abstract

Abstract A series of amino acid modifying reagents were tested for their effects on the activity of the (1→3)-β-glucan synthase (EC 2.4.1.34) from endosperm of ryegrass ( Lolium multiflorum ). Of these reagents only the carbodiimide EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), DEPC (diethyl pyrocarbonate), iodine, Cu 2+ , Hg 2+ , phenylmercuric nitrate, N -bromosuccinimide and 2-hydroxy-5-nitrobenzylbromide gave significant inhibition. It was concluded that carboxyl and His groups are likely to be essential for enzyme activity but the involvement of Trp residues was not excluded. Significantly, free thiols or disulfides do not appear to be necessary for enzyme activity. The kinetics of the inhibition reaction with diethyl pyrocarbonate were complex. No evidence for methoxyformylation of His was obtained by measurements of absorbance at 242 nm. EDC inhibited in a time and concentration dependent manner, with kinetics suggesting that no reversible complex between the enzyme and the EDC was formed. The apparent bimolecular rate constant was 0.029 M −1 s −1 . The order of the inactivation was 1 indicating that one molecule of EDC is bound per active site. The rate of inhibition is slowed in the presence of UDP–Glc, suggesting that the modified groups may be located in or near the substrate-binding domain. The pH dependence of the rate of inactivation indicates the presence of a critical group with a p K a of 5.7. These results are consistent with the finding that a `D,D,D(35)Q(RQ)XRW' motif is present in a number of repetitive β-glycan synthases and suggest that the critical acidic group may be an aspartate.