title: Substrate preference of a Geobacillus maltogenic amylase: a kinetic and thermodynamic analysis.
abstract: The gene encoding a maltogenic amylase (MAase) from a newly isolated strain of thermophilic Geobacillus has been isolated, cloned and expressed. Following purification, biochemical and structural characterization have been performed. The enzyme exhibited maximal activity at a broad temperature range between 55 and 65 °C, clearly higher than that of other dimeric MAses. The optimum pH was 6.0 and catalytic activity increased by of Li(+) and K(+). A noticeable preference was demonstrated for α-, β- and γ-cyclodextrin (CD) compared to polymeric substrates (amylose, amylopectin, glycogen and starch) possibly due to steric interference. The affinity for CD substrates increased in the order of γ-CD>β-CD>α-CD, but k(cat)/K(m) increased as α-CD>β-CD>γ-CD, implying that increased substrate specificities are mainly attribute to kcat. Thermodynamic analysis of the activation process showed that improved activity (decrease in ΔG(#)) is accompanied by increases in activation entropy (ΔS(#)) for aforementioned substrates. Molecular docking on the binding interactions between substrates and active site residues revealed a considerably higher accessible surface area for the active site residues in the presence of α-CD than β-CD, indicating that interactions in the transition state are stronger in the presence of α-CD. This result explains the increased ΔH(#) of the activation process and increased K(m) of the enzyme in the presence of α-CD, compared to that of β-CD. This study, which presents the first detailed comparative analysis on the substrate preference of dimeric MAases for different substrates, may shed some lights into the molecular mechanism of these enzymes.
authors: Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Iran.
authors: Samira
authors: S
authors: Nasrollahi
authors: Leila
authors: L
authors: Golalizadeh
authors: Reza H
authors: RH
authors: Sajedi
authors: Majid
authors: M
authors: Taghdir
authors: S Mohsen
authors: SM
authors: Asghari
authors: Mehdi
authors: M
authors: Rassa
mesh_terms: D000595:Amino Acid Sequence; D056506:Geobacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D007700:Kinetics; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D011487:Protein Conformation; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D007477:Ions; D008670:Metals; D012336:RNA, Ribosomal, 16S; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C120909:glucan 1,4-alpha-maltohydrolase
keywords: Cyclodextrin; Geobacillus; Kinetic and thermodynamic analysis; Maltogenic amylase; Substrate specificity