TY - JOUR
T1 - Both binding sites of the starch-binding domain of Aspergillus niger glucoamylase are essential for inducing a conformational change in amylose
AU - Giardina, Thierry
AU - Gunning, A. Patrick
AU - Juge, Nathalie
AU - Faulds, Craig B.
AU - Furniss, Caroline S.M.
AU - Svensson, Birte
AU - Morris, Victor J.
AU - Williamson, Gary
PY - 2001/11/9
Y1 - 2001/11/9
N2 - The interaction of the two binding sites of the starch-binding domain (SBD) of Aspergillus niger glucoamylase 1 (GA-I) with substrate has been investigated by using atomic force microscopy (AFM) and UV difference spectroscopy in combination with site-specific mutants of both SBD and GA-I. The SBD possesses two binding sites with distinct affinities towards the soluble linear substrate maltoheptaose; dissociation constants (Kd) of 17 and 0.95 μM were obtained for W563 K (binding site 2 mutant) and W590 K (binding site 1 mutant), respectively, compared to an apparent Kd of 23 μM for the wild-type SBD. Further, the two sites are almost but not totally independent of each other for binding, since abolishing one site does not prevent the amylose chain binding to the other site. Using AFM, we show that the amylose chains undergo a conformational change to form loops upon binding to the SBD, using either the recombinant wild-type SBD or a catalytically inactive mutant of GA-I. This characteristic conformation of amylose is lost when one of the SBD binding sites is eliminated by site-directed mutagenesis, as seen with the mutants W563 K or W590 K. Therefore, although each binding site is capable of simple binding to a ligand, both sites must be functional in order to induce a gross conformational change of the amylose molecules. Taken together these data suggest that for the complex with soluble amylose, SBD binds to a single amylose chain, site 1 being responsible for the initial recognition of the chain and site 2 being involved in tighter binding, leading to the circularisation of the amylose chain observed by AFM. Binding of the SBD to the amylose chain results in a novel two-turn helical amylose complex structure. The binding of parallel amylosic chains to the SBD may provide a basis for understanding the role of the SBD in facilitating enzymatic degradation of crystalline starches by glucoamylase 1.
AB - The interaction of the two binding sites of the starch-binding domain (SBD) of Aspergillus niger glucoamylase 1 (GA-I) with substrate has been investigated by using atomic force microscopy (AFM) and UV difference spectroscopy in combination with site-specific mutants of both SBD and GA-I. The SBD possesses two binding sites with distinct affinities towards the soluble linear substrate maltoheptaose; dissociation constants (Kd) of 17 and 0.95 μM were obtained for W563 K (binding site 2 mutant) and W590 K (binding site 1 mutant), respectively, compared to an apparent Kd of 23 μM for the wild-type SBD. Further, the two sites are almost but not totally independent of each other for binding, since abolishing one site does not prevent the amylose chain binding to the other site. Using AFM, we show that the amylose chains undergo a conformational change to form loops upon binding to the SBD, using either the recombinant wild-type SBD or a catalytically inactive mutant of GA-I. This characteristic conformation of amylose is lost when one of the SBD binding sites is eliminated by site-directed mutagenesis, as seen with the mutants W563 K or W590 K. Therefore, although each binding site is capable of simple binding to a ligand, both sites must be functional in order to induce a gross conformational change of the amylose molecules. Taken together these data suggest that for the complex with soluble amylose, SBD binds to a single amylose chain, site 1 being responsible for the initial recognition of the chain and site 2 being involved in tighter binding, leading to the circularisation of the amylose chain observed by AFM. Binding of the SBD to the amylose chain results in a novel two-turn helical amylose complex structure. The binding of parallel amylosic chains to the SBD may provide a basis for understanding the role of the SBD in facilitating enzymatic degradation of crystalline starches by glucoamylase 1.
KW - Aspergillus niger
KW - Atomic force microscopy
KW - Glucoamylase
KW - Starch-binding domain
KW - UV difference spectroscopy
UR - http://www.scopus.com/inward/record.url?scp=0035834494&partnerID=8YFLogxK
U2 - 10.1006/jmbi.2001.5097
DO - 10.1006/jmbi.2001.5097
M3 - Article
C2 - 11700070
AN - SCOPUS:0035834494
SN - 0022-2836
VL - 313
SP - 1149
EP - 1159
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 5
ER -