Alpha-glucan binding of potato-tuber starch-branching enzyme I as determined by tryptophan fluorescence quenching, affinity electrophoresis and steady-state kinetics.
The affinity of potato tuber starch-branching enzyme-I (PSBE-I) for various linear malto-oligosaccharides, cyclodextrins, (CDs) and macromolecular alpha-glucans was investigated by alpha-glucan induced fluorescence quenching of intrinsic PSBE-I tryptophan residues and by affinity electrophoresis. alpha-Glucan binding was characterised by distinct shifts towards shorter wavelengths of the PSBE-I fluorescence emission spectrum and by concomitant reductions in fluorescence intensity. The magnitudes of both the maximum shift in emission spectrum and reduction in fluorescence intensity were dependent on the alpha-glucan ligands used. Maximum Kd for a range of linear malto-oligosaccharides analysed was 0.13 mM as found at a degree of polymerisation (DP) of 13. Large differences in dissociation constants were measured for CDs with DP 6 (alpha-CD, 6.0 mM), DP 7 (beta-CD, 0.25 mM) and DP 8 (gamma-CD, 0.67 microM). The high-molecular-mass alpha-glucans amylose and amylopectin, both substrates for PSBE-I, showed apparent affinities of 0.018 and 0.066 mg/ml, respectively. Small linear and cyclic oligosaccharides competed with amylopectin in the affinity electrophoresis system and they were also competitive inhibitors for PSBE-I activity. The affinities for oligosaccharides as measured by competition were, however, about 10-fold lower than as measured by fluorescence quenching suggesting the existence of a separate oligosaccharide binding site on PSBE-I. Affinity electrophoresis revealed multiform heterogeneity in the enzyme preparation with respect to alpha-glucan interaction.
Blennow
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Cooperative Research Centre for Plant Science, Canberra, Australia.
Viksø-Nielsen
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Morell
M K
MK
D015061:1,4-alpha-Glucan Branching Enzyme / Q000378:metabolism*; D000687:Amylopectin / Q000378:metabolism; D000688:Amylose / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D003505:Cyclodextrins / Q000378:metabolism; D005936:Glucans / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D009844:Oligosaccharides / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism*; D011485:Protein Binding; D011198:Solanum tuberosum / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D003505:Cyclodextrins; D005936:Glucans; D009844:Oligosaccharides; D010940:Plant Proteins; C016549:maltohexaose; D014364:Tryptophan; D000688:Amylose; D000687:Amylopectin; D015061:1,4-alpha-Glucan Branching Enzyme