RESTORATION OF CATALYTIC ACTIVITY BEYOND WILD-TYPE LEVEL IN GLUCOAMYLASE FROM ASPERGILLUS-AWAMORI BY OXIDATION OF THE GLU400-]CYS CATALYTIC-BASE MUTANT TO CYSTEINESULFINIC ACID

Glucoamylase catalyzes the hydrolysis of glucosidic bonds with inversi on of the anomeric configuration. Site-directed mutagenesis and three- dimensional structure determination of the glucoamylase from Aspergill us awamori previously identified Glu179 and Glu400 as the general acid and base catalyst, respectively. The average distance between the two carboxyl groups was measured to be 9.2 Angstrom, which is typical for inverting glycosyl hydrolases. In the present study, this distance wa s increased by replacing the catalytic base Glu400 with cysteine which was then oxidized to cysteinesulfinic acid. Initially, this oxidation occurred during attempts to carboxyalkylate the Cys400 residue with i odoacetic acid, 3-iodopropionic acid, or 4-bromobutyric acid. However, endoproteinase Lys-C digestion of modified glucoamylase followed by h igh-pressure liquid chromatography in combination with matrix assisted laser desorption ionization/time-of-flight mass spectrometry on purif ied peptide fragments demonstrated that all enzyme derivatives contain ed the cysteinesulfinic acid oxidation product of Cys400. Subsequently , it was demonstrated that treatment of Glu400-->Cys glucoamylase with potassium iodide in the presence of bromine resulted in complete conv ersion to the cysteinesulfinic acid product. As expected, the catalyti c base mutant Glu400-->Cys glucoamylase had very low activity, i.e., 0 .2% compared to wild-type. The oxidation of Cys400 to cysteinesulfinic acid, however, restored activity (k(cat)) on alpha-1,4-linked substra tes to levels up to 160% of the wild-type glucoamylase which correspon ded to approximately a 700-fold increase in the k(cat) of the Glu400-- >Cys mutant glucoamylase. Whereas G1u400-->Cys glucoamylase was much l ess thermostable and more sensitive to guanidinium chloride than the w ild-type enzyme, the oxidation to cysteinesulfinic acid was accompanie d by partial recovery of the stability.