Identification of important amino acid residues of amylomaltases through chemical modification technique

Abstract

The aim of this study is to compare and identify important amino acid residues in two bacterial amylomaltases (AMs): CGAM from Corynebacterium glutamicum and THAM from AM gene screened from soil DNA, and a plant MeDPE1 from cassava Manihot esculenta Crantz tuber using chemical modification method. The purified AMs were treated with various modifying reagents specific for different functional groups. For all AMs, almost total activity loss was observed with N-bromosuccinamide (NBS) modification. The activity band on native-PAGE of CGAM was disappeared after treatment with NBS or Succinic anhydride (SAH), while a small decrease in migration was observed after modification by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). These results suggest the importance of Trp, Asp/Glu and Lys for AMs activity. Substrate protection using maltotriose (G3) suggests that these residues are at or around active site region of these AMs, except for Lys in MeDPE1. Then, the effect of modification of these residues on AMs’ properties was analyzed. The inactivation of AMs by these reagents was performed at IC₅₀ concentrations at pH 6.0. From the pseudo-first order kinetic constant of the inactivation reaction, MeDPE1 was most sensitive to all reagents except for NBS while THAM was the least sensitive. pI of native and NBS-modified AMs were similar with 6.9 and 5.8 for CGAM and THAM, respectively. Native and NBS-modified of all AMs prefer G3 in disproportionation but G1 in starch transglucosylation reaction. The kcat/Km values of the modified CGAM were significantly decreased in both reactions. The Ki values of acarbose inhibitor for the modified enzyme were increased. The number of modified Trp in the active site was about one and three residues for CGAM and THAM, respectively. The secondary structure of CGAM and the Trp environment showed a slight change after NBS treatment. The product pattern of linear oligosaccharides and LR-CDs was not much affected by Trp modification. The major LR-CDs from native and modified CGAM was CD24-CD32 with CD27-CD28 as maximum while THAM yielded CD22-CD28 with CD23-CD24 maximum product.