Enzymatic properties of the cysteinesulfinic acid derivative of the catalytic-base mutant Glu400-->Cys of glucoamylase from Aspergillus awamori.
The pKa of the catalytic base was lowered and its distance to the general acid catalyst, Glu179, was increased in the glucoamylase from Aspergillus awamori by replacing the catalytic base Glu400 with cysteine followed by oxidation to cysteinesulfinic acid [Fierobe, H.-P., Mirgorodskaya, E., McGuire, K. A., Roepstorff, P., Svensson, B. and Clarke, A. J. (1998) Biochemistry 37, 3743-3752. 1H NMR spectroscopy demonstrated that the oxidized mutant Glu400-->Cys-SO2H glucoamylase, like the wild-type, catalyzed hydrolysis with inversion of the anomeric configuration of the product. Relative to the catalytic base mutant Glu400-->Cys, the Cys400-SO2H glucoamylase had 700 times higher kcat toward maltose, while K(m) was unchanged. Compared to wild-type glucoamylase, the Cys400-SO2H derivative had kcat values of 150-190% and 85-320% on malto- and isomaltooligosaccharides, respectively, while K(m) values were similar to those of wild-type with the two disaccharides and 3.5-5.5- and 1.8-2.5-fold higher for the longer malto- and isomaltooligosaccharides substrates, respectively. The pH-activity dependence at saturating concentration of maltose indicated that the pKa of the catalytic base Cys400-SO2H was about 0.5 pH unit lower than that of wild-type Glu400. The Ki of Cys400-SO2H glucoamylase for the pseudotetrasaccharide and potent inhibitor acarbose increased more than 10(4)-fold, but Ki values of the mono- and disaccharide analogues 1-deoxynojirimycin and beta-O-methylacarviosinide were unchanged, suggesting perturbation at binding subsites beyond the catalytic center. A distinct property of Cys400-SO2H glucoamylase was the catalysis of the condensation of beta-D-glucopyranosyl fluoride and subsequent hydrolysis of the product to beta-glucose, under conditions where this was not detected for the wild-type enzyme.
Fierobe
H P
HP
Department of Chemistry, Carlsberg Laboratory, Copenhagen Valby, Denmark.
Clarke
A J
AJ
Tull
D
D
Svensson
B
B
D017485:1-Deoxynojirimycin / Q000378:metabolism / Q000494:pharmacology; D020909:Acarbose; D019943:Amino Acid Substitution / Q000235:genetics*; D000606:Amino Sugars / Q000378:metabolism / Q000494:pharmacology; D001230:Aspergillus / Q000201:enzymology*; D001667:Binding, Competitive; D002384:Catalysis; D003545:Cysteine / Q000031:analogs & derivatives* / Q000235:genetics* / Q000378:metabolism; D004789:Enzyme Activation; D005087:Glucan 1,4-alpha-Glucosidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005947:Glucose / Q000031:analogs & derivatives / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics; D006836:Hydro-Lyases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007534:Isomaltose / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D018377:Neurotransmitter Agents; D009844:Oligosaccharides / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D014312:Trisaccharides / Q000378:metabolism / Q000494:pharmacology
D000606:Amino Sugars; D018377:Neurotransmitter Agents; D009844:Oligosaccharides; D011134:Polysaccharides; D014312:Trisaccharides; C021705:maltooligosaccharides; C076099:acarviosine; D017485:1-Deoxynojirimycin; C030364:glucosyl fluoride; D018698:Glutamic Acid; D007534:Isomaltose; D005087:Glucan 1,4-alpha-Glucosidase; D006836:Hydro-Lyases; D005947:Glucose; D003545:Cysteine; D020909:Acarbose; C013461:cysteine sulfinic acid