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 inversion of the anomeric configuration. Site-directed mutagenesis and three-dimensional structure determination of the glucoamylase from Aspergillus 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 A, which is typical for inverting glycosyl hydrolases. In the present study, this distance was 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 iodoacetic acid, 3-iodopropionic acid, or 4-bromobutyric acid. However, endoproteinase Lys-C digestion of modified glucoamylase followed by high-pressure liquid chromatography in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry on purified peptide fragments demonstrated that all enzyme derivatives contained 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 conversion to the cysteinesulfinic acid product. As expected, the catalytic 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 (kcat) on alpha-1,4-linked substrates to levels up to 160% of the wild-type glucoamylase which corresponded to approximately a 700-fold increase in the kcat of the Glu400-->Cys mutant glucoamylase. Whereas Glu400-->Cys glucoamylase was much less thermostable and more sensitive to guanidinium chloride than the wild-type enzyme, the oxidation to cysteinesulfinic acid was accompanied by partial recovery of the stability.
Fierobe
H P
HP
Department of Chemistry, Carlsberg Laboratory, Copenhagen Valby, Denmark.
Mirgorodskaya
E
E
McGuire
K A
KA
Roepstorff
P
P
Svensson
B
B
Clarke
A J
AJ
D000478:Alkylation; D019943:Amino Acid Substitution / Q000235:genetics*; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D002264:Carboxylic Acids / Q000378:metabolism; D002384:Catalysis; D003545:Cysteine / Q000031:analogs & derivatives* / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D005087:Glucan 1,4-alpha-Glucosidase / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics; D007525:Isoelectric Focusing; D009154:Mutation; D018377:Neurotransmitter Agents; D010084:Oxidation-Reduction
D002264:Carboxylic Acids; D018377:Neurotransmitter Agents; D018698:Glutamic Acid; D005087:Glucan 1,4-alpha-Glucosidase; D003545:Cysteine; C013461:cysteine sulfinic acid