Enzyme kinetics and chemical modification of alpha-1,4-glucan lyase from Gracilariopsis sp.

The kinetic properties and active site amino acids of alpha-1,4-glucan lyas e from the marine red macroalga Glacilariopsis sp. were examined. Using H-1 NMR spectroscopy the alpha-1,4-glucan lyase was found to degrade alpha- an d beta-maltose at different rates. The effect of pH on the kinetic constant s suggested the presence of two catalytically important amino acids in the active site with pK(a) values of 3.5 and 6.2. The former indicated the pres ence of an ionised aspartate or glutamate residue in the active site. This was tested using the carboxyl specific reagent EDAC, which inhibited enzyme activity in a time dependent manner when an external nucleophile was added . No protection against the inactivation was obtained by addition of amylop ectin, maltitol or 1-deoxinojirimycin. Inactivation decreased V-max over 2. 5-fold with little effect on K-m which supports the direct involvement of a carboxyl group in catalysis. (C) 2000 Elsevier Science Ltd. All rights res erved.