Enzyme kinetics and chemical modification of alpha-1,4-glucan lyase from Gracilariopsis sp
2000
Nyvall, P. | Pedersen, M. | Kenne, L. | Gacesa, P.
The kinetic properties and active site amino acids of alpha-1,4-glucan lyase from the marine red macroalga Gracilariopsis sp. were examined. Using 1H NMR spectroscopy the alpha-1,4-glucan lyase was found to degrade alpha-and-beta-maltose at different rates. The effect of pH on the kinetic constants suggested the presence of two catalytically important amino acids in the active site with pKa values of 3.5 and 6.2. The former indicated the presence 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 amylopectin, maltitol or 1-deoxinojirimycin. Inactivation decreased Vmax over 2.5-fold with little effect on Km which supports the direct involvement of a carboxyl group in catalysis.
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