SCANNING-TUNNELING-MICROSCOPY OF ASPERGILLUS-NIGER GLUCOAMYLASES

The primary structure of glucoamylase 1 (glucan-1,4-alpha-glucosidase, EC 3.2.1.3) from Aspergillus niger consists of a catalytic and a binding domain separated by an 0-glycosylated peptide 40 amino acid residues in length. Scanning tunnelling microscopic (STM) images of glucoamylase 2 (the catalytic domain with linker peptide) and a proteolytically cleaved product (the binding domain GlC, residues 471-616 of glucoamylase 1) confirmed that these proteins are globular and nearly spherical. Observed dimensions are: isolated catalytic domain (median half-height diameter, d(m) = 5.8 nm, in-plane axial ratio, gamma = 1.15 : 1), isolated binding domain (d(m) = 2.2 nm, gamma = 1.18 : 1), catalytic domain within glucoamylase 1 (d(m) = 5.9 nm, gamma = 1.1 : 1) and binding domain within glucoamylase 1 (d(m) = 3.4 nm, gamma = 1.1 : 1) However, STM images of glucoamylase 1 suggest that the enzyme consists of two spatially separated globular domains. Measurements of the inter-domain spacing (median spacing between domain centres in glucoamylase 1, d(s) = 9.5 nm) suggest that the linker glycopeptide is extended and semi-rigid. These data suggest that the 0-glycosylated sequence may play an important role in determining the shape and functionality of the enzyme.