IDENTIFICATION OF CARBOXYLIC-ACID RESIDUES IN GLUCOAMYLASE-G2 FROM ASPERGILLUS-NIGER THAT PARTICIPATE IN CATALYSIS AND SUBSTRATE BINDING

Functionally important carboxyl groups in glucoamylase G2 form 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 phenylthiohydantions. 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 invariant in fungal glucoamylases. Glu180 was tentatively identified as a catalytic group on the basis of sequence alignments to catalytic regions in isomaltase and α‐amylase. The partially radiolabelled Asp112 corresponds in Taka‐amylase A to Tyr75 situated in a substrate binding loop at a distance from the site of cleavage. A possible correlation between carbodiimide modification of an essential carboxyl group and its role in the glucoamylase catalysis is discussed. Copyright © 1990, Wiley Blackwell. All rights reserved