Summary
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1.
The basic concept and outline of the subsite theory were described, which correlates quantitatively the subsite structure (the arrangement of subsite affinities) to the action pattern of amylases in a unified manner.
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2.
The subsite structures of several amylases including glucoamylase were summarized.
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3.
In parallel with the theoretical prediction obtained therefrom, the binding subsites of glucose, gluconolactone and linear substrates to Rhizopus glucoamylase were investigated experimentally, by using steady-state inhibition kinetics, difference absorption spectrophotometry, and fluorometric titration.
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4.
From several lines of evidence, it was concluded that gluconolactone, a transition state analogue, is bound at Subsite I (nonreducing end side) where a tryptophan residue is located.
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5.
The stopped-flow kinetic studies have revealed that all the ligand bindings studied consist of two-step mechanism in which a bimolecular association between the enzyme and a ligand to form a loosely bound complex (EL) followed by the unimolecular isomerization process in which EL converts to the final firmly bound EL* complex. For substrates the EL* may be the productive complex and the fluorescence of the tryptophan located at Subsite 1 is quenched in their isomerization process, most probably a relocation of ligand to occupy this subsite.
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Hiromi, K., Ohnishi, M. & Tanaka, A. Subsite structure and ligand binding mechanism of glucoamylase. Mol Cell Biochem 51, 79–95 (1983). https://doi.org/10.1007/BF00215589
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DOI: https://doi.org/10.1007/BF00215589