Chemical modification of carboxyl groups in glucoamylase from Aspergillus niger

Abstract

Glucoamylases GI and G2 from Aspergillus niger lost the catalytic activity by prolonged treatment with 1 -ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide (EAC). A tolal of 23 and 16 carboxyl groups were substituted out of 71 and 54 present in the GI and G2 enzyme, respectively. The kinetics and the pH-dependence of the inactivation were compatible with the existence of one essential carboxyl group with a pK~ 5.5. The inhibitor acarbose (a pseudotetrasaccharide) and the substrate maltose prevented EAC-modification of 4 and 2 carboxyl groups, respectively, with considerable retention of enzyme activity. Following removal of these fig,ands, differenlial labelling of critical carboxyl residue(s) was achieved by means of [3H]EAC.Glucoamylase G2 with 12 EAC groups incorporated, prepared in the presence of acarbose, displayed a two-fold increase of K~ and a slight reduction of V,~ for hydrolysis of starch relative to the unmodified enzyme. Both K~ and Vm~ for hydrolysis of maltose were almost unaffected. In addition, maltose and three inhibitors perturbed the UV absorbance spectrum of this derivative. An altered shape of the acarbose-induced difference spectrum implied substitution of a carboxyl group near a binding tryptophanyl residue at a distance from the catalytic site. Efficient protection by acarbose against only the second part of the biphasic course of inactivation similarly reflected that residues playing different roles in the mechanism of action reacted with EAC.In separate experiments, evidence was obtained for a preferred reaction of ethylenimine with enzymatically important carboxyl groups in glucoamylase.

Side chain reactivities of glucoamylase G2 from aspergillus niger evaluated by group-specific chemical modifications

“…Various reagents in large excesses were employed to modify lysyl, arginyl, tyrosyl, and methionyl residues, but only the carboxyl groups and tryptophanyl residues as found earlier seemed essential for activity and thermostability (Table IV) (8,9,42,46).…”

“…Enzymatically active N-bromosuccinimide oxidized or carbodiimide modified G2 were prepared in the presence of acarbose as earlier described (8,9,42,46).…”

“…The two forms are equally active towards soluble substrates, but only G 1 is able to degrade raw starch (47). A number of tryptophanyl residues and carboxyl groups involved in substrate binding and catalysis have been identified by chemical modification and sequencing (8,9,42,46). The related enzymes, a-amylase and ct-glucosidase, m adchtlon possess critical histidyl residues (2, 6,11,13,14,19,23,38), but modification of glucoamylase by diethyl pyrocarbonate ( 18,31,35) has only been found to decrease the affinity for soluble starch (18).…”

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

“…niger glucoamylase [5]. The glucoamylase G2 (40 mg, 0.55 pmol) in 0.05 M Mes pH 6.0 (10 ml) was first treated with EAC (0.1 M) in the presence of either acarbose (580 pM) or maltose (0.6 M) for 20 rnin at room temperature.…”

“…We used the strong inhibitor acarbose [28] to protect glucoamylase against inactivation by carbodiimide. When the acarbose was removed, the essential carboxyl groups were reacted with radioactive carbodiimide [5]. Sequencing identified the differentially labelled positions in a highly conserved part of the enzyme [29] which resembles catalytic regions in both isomaltase [17, 181 and a-amylase [8, 131.…”