- Synthesis and characterisation of novel chromogenic substrates for human pancreatic α-amylase
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Derivatives of maltose and maltotriose were chemically synthesised as substrates for human pancreatic α-amylases and subjected to kinetic analysis. Rates measured were shown to reflect both hydrolysis and transglycosylation reactions. 4-O-Methylated derivatives of these substrates underwent only hydrolysis, thereby simplifying kinetic analyses. These modified substrates may be used for the detection and kinetic analysis of α-amylases, and are useful in rapidly screening for novel α-amylase inhibitors and for subsequent kinetic characterisation.
- Damager, Iben,Numao, Shin,Chen, Hongming,Brayer, Gary D.,Withers, Stephen G.
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p. 1727 - 1737
(2007/10/03)
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- Study of the action of human salivary alpha-amylase on 2-chloro-4-nitrophenyl α-maltotrioside in the presence of potassium thiocyanate
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The degradation mechanism of a synthetic substrate, 2-chloro-4-nitrophenyl α-maltotrioside (CNP-G3), by human salivary alpha-amylase (HSA) was investigated by kinetic and product analyses. It was observed that the enzyme attacked the various CNP-maltooligosaccharides (CNP-G3, to CNP-G6) releasing free CNP. Addition of 500 mM potassium thiocyanate (KSCN) was also found to greatly increase the rates of CNP-release. It was the fastest with CNP-G3, and, in the presence of KSCN, was almost comparable to that of degradation of maltopentaose (G5). On the other hand, addition of KSCN decreased the rate of cleavage between glucan-glucan bonds in maltopentaose. Product analysis showed that KSCN addition altered the cleavage distribution which occurred 100% at the bond between CNP and G3, and that product distribution of free CNP was largely dependent on substrate concentration. Formation of CNP-G6, a larger product than the original substrate CNP-G3, was found to be present in the digest at high concentrations of substrate and in the presence of KSCN. Based on these results, a degradation pathway for CNP-G3 involving transglycosylation besides direct hydrolysis is proposed. The increase of the CNP-release by the addition of KSCN would result from a corresponding increase in the interaction between the CNP moiety and the corresponding subsite near the catalytic site, as well as the enhancement of the catalytic efficiency.
- Suganuma, Toshihiko,Maeda, Yoshiaki,Kitahara, Kanefumi,Nagahama, Tomonori
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p. 219 - 227
(2007/10/03)
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