Studies on the subsite structure of amylases.  I. Interaction of glucoamylase with substrate and analogues studied by difference-spectrophotometry.
Studies were made on the ultraviolet difference-spectra of glucoamylase from Rhizopus niveus [EC 3.2.1.3] specifically produced by the substrate maltose and the inhibitors, glucose, glucono-1: 5-lactone (gluconolactone), methyl beta-D-glucoside, cellubiose, and cyclohexa-, and cyclohepta-amyloses.  Of these, maltose and gluconolactone produced characteristic difference spectra with a trough near 300 nm.  Based on studies with a model compound for a tryptophan residue, Ac-Trp, this trough was attributed to the effect of a negative charge upon the tryptophan residue.  From the concentration dependency of the difference spectra, the dissociation constants of the complexes between the enzyme and maltose, glucose, and gluconolactone were evaluated to be 1.2 mM, 51 mM, and 1.5 mM, respectively.  These values are in good agreement with the values of Km or K1 obtained from the steady-state kinetics.  The difference-spectrophotometric data suggested that referring to the values of subsite affinities of glucoamylase, maltose, and gluconolactone occupy mainly Subsite 1, where the non-reducing-end glucose residue of a substrate is bound in a productive form and that a tryptophan residue with shows a trough near 300 nm in difference spectra is located in this subsite.
D000596:Amino Acids / Q000032:analysis; D000688:Amylose / Q000031:analogs & derivatives / Q000378:metabolism; D003508:Cycloheptanes; D003510:Cyclohexanes; D005942:Gluconates; D005947:Glucose / Q000378:metabolism; D005959:Glucosidases / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism; D007700:Kinetics; D008320:Maltose / Q000378:metabolism; D011134:Polysaccharides; D012233:Rhizopus / Q000201:enzymology; D013053:Spectrophotometry / Q000379:methods; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000378:metabolism
D000596:Amino Acids; D003508:Cycloheptanes; D003510:Cyclohexanes; D005942:Gluconates; D005960:Glucosides; D011134:Polysaccharides; D008320:Maltose; D014364:Tryptophan; D000688:Amylose; D005959:Glucosidases; D005947:Glucose