PMID: 2108020Feb 22, 1990Paper

Identification of carboxylic acid residues in glucoamylase G2 from Aspergillus niger that participate in catalysis and substrate binding

European Journal of Biochemistry
B SvenssonH Møller

Abstract

Functionally important carboxyl groups in glucoamylase G2 from Aspergillus niger were identified using a differential labelling approach which involved modification of the acarbose-inhibited enzyme with 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC) and inactivation by [3H]EAC following removal of acarbose. Subsequent sequence localization of the substituted acidic residues was facilitated by specific phenylthiohydantoins. The acid cluster Asp176, Glu179 and Glu180 reacted exclusively with [3H]EAC, while Asp112, Asp153, Glu259 and Glu389 had incorporated both [3H]EAC and EAC. It is conceivable that one or two of the [3H]EAC-labelled side chains act in catalysis while the other fully protected residue(s) participates in substrate binding probably together with the partially protected ones. Twelve carboxyl groups that reacted with EAC in the enzyme-acarbose complex were also identified. Asp176, Glu179 and Glu180 are all invariant in fungal glucoamylases. Glu180 was tentatively identified as a catalytic group on the basis of sequence alignments to catalytic regions in isomaltase and alpha-amylase. The partially radiolabelled Asp112 corresponds in Taka-amylase A to Tyr75 situated in a substrate binding loop at a distance...Continue Reading

References

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Citations

Jan 11, 2001·Biochimica Et Biophysica Acta·J SauerB Svensson
Jan 17, 2003·Journal of Biomolecular Structure & Dynamics·Hsuan-Liang LiuChia-Ming Hsu
Jul 15, 1992·European Journal of Biochemistry·G WilliamsonM P Williamson
Feb 15, 1997·European Journal of Biochemistry·B HülsewehC P Hollenberg
Jun 29, 2000·Annual Review of Biochemistry·H D Ly, S G Withers
Feb 1, 1994·European Journal of Biochemistry·M R Bray, A J Clarke
May 29, 2001·The Journal of Biological Chemistry·A NakayamaS Tabata
Apr 7, 1995·The Journal of Biological Chemistry·L ChenP B Høj
Oct 20, 2021·The FEBS Journal·Takayoshi TagamiAtsuo Kimura

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