Evaluation of subsite affinities of soybean .BETA.-amylase by product analysis.

Suganuma, Toshihiko; Ohnishi, Masatake; Kitano, Hiromi; Morita, Yuhei

doi:10.1271/bbb1961.44.1111

Cited by 15 publications

(10 citation statements)

References 2 publications

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“…By combining the docking results from this study with previously ascertained facts, we can gain insight into the mechanics of ␤-amylase's catalytic action. Subsite Ϫ2 has the highest substrate binding affinity, as Suganuma et al 7 found experimentally. Our docking experiments confirm this but additionally suggest that subsite ϩ1 may also have a relatively high binding affinity, indicated by the energetically favorable ␣-maltotriose dockings in subsites ϩ1,ϩ2,ϩ3.…”

Section: Docking Of Trisaccharides In the Open Conformationmentioning

confidence: 55%

“…However, docking in this mode is not ideal, and we found that the proteinligand interactions were different from the ones docked in subsites Ϫ2,Ϫ1,ϩ1 and ϩ1,ϩ2,ϩ3. Using the cleavage distribution of maltotriose, Suganuma et al 7 showed that subsite Ϫ2 had the highest binding affinity of subsites Ϫ2, ϩ2, and ϩ3, but their methodology did not allow them to calculate the affinities of the two subsites surrounding the cleavage point.…”

Section: Docking Of ␣-Maltotriose In the Closed Conformationmentioning

confidence: 99%

“…Since the rate of oligosaccharide hydrolysis increases with each additional glucosyl unit up to five, 7 it can be inferred that a fifth subsite exists. Subsite Ϫ2 has the highest binding affinity for a glucosyl ring.…”

mentioning

confidence: 99%

“…Subsite Ϫ2 has the highest binding affinity for a glucosyl ring. 7 The enzyme's hydrolytic site is located between subsites Ϫ1 and ϩ1. It consists of two highly conserved residues, Glu186 and Glu380, located below and above, respectively, the plane of the second glucosyl unit from the nonreducing end of the chain (Glc B ).…”

mentioning

confidence: 99%

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Automated docking of ?-(1?4)- and ?-(1?6)-linked glucosyl trisaccharides and maltopentaose into the soybean ?-amylase active site

2000

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The Lamarckian genetic algorithm of AutoDock 3.0 was used to dock alpha-maltotriose, methyl alpha-panoside, methyl alpha-isopanoside, methyl alpha-isomaltotrioside, methyl alpha-(6(1)-alpha-glucopyranosyl)-maltoside, and alpha-maltopentaose into the closed and, except for alpha-maltopentaose, into the open conformation of the soybean beta-amylase active site. In the closed conformation, the hinged flap at the mouth of the active site closes over the substrate. The nonreducing end of alpha-maltotriose docks preferentially to subsites -2 or +1, the latter yielding nonproductive binding. Some ligands dock into less optimal conformations with the nonreducing end at subsite -1. The reducing-end glucosyl residue of nonproductively-bound alpha-maltotriose is close to residue Gln194, which likely contributes to binding to subsite +3. In the open conformation, the substrate hydrogen-bonds with several residues of the open flap. When the flap closes, the substrate productively docks if the nonreducing end is near subsites -2 or -1. Trisaccharides with alpha-(1-->6) bonds do not successfully dock except for methyl alpha-isopanoside, whose first and second glucosyl rings dock exceptionally well into subsites -2 and -1. The alpha-(1-->6) bond between the second and third glucosyl units causes the latter to be improperly positioned into subsite +1; the fact that isopanose is not a substrate of beta-amylase indicates that binding to this subsite is critical for hydrolysis.

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Section: Docking Of Trisaccharides In the Open Conformationmentioning

confidence: 55%

Section: Docking Of ␣-Maltotriose In the Closed Conformationmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Automated docking of ?-(1?4)- and ?-(1?6)-linked glucosyl trisaccharides and maltopentaose into the soybean ?-amylase active site

2000

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“…This mode of action can be assumed to repeat rapidly and, even though the DP of the D-glucopyranosyl polymer reaches a size at which the inclusion complex with Leu383 cannot be formed in the course of the enzymic chewing, it may still continue because the force of the hydrophobic interactions can retain the polymer in the Leu383 region. From the observation that the K,,, value for maltotriose increases far beyond the value for single-stranded amylose [34,351, it may be the case that P-amylase cannot retain its substrate any longer in the above region.…”

Section: Discussionmentioning

confidence: 99%

Functional Analysis of Glu380 and Leu383 of Soybean β‐Amylase

Totsuka¹,

Fukazawa²

1996

European Journal of Biochemistry

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Soybean P-amylase, comprising a @/a),-barrel core with a mobile loop, similar to that of triose phosphate isomerase, was mutated by site-directed mutagenesis at residues Glu380 and Leu383. X-ray crystallographic findings suggest that Glu380 is the counterpart of the catalytic site (Glu186) and that Leu383, located near the active-site cavity, forms an inclusion complex with cyclomaltohexaose. Separate substitutions of Glu380 by Gln and Asp completely eliminated the activity without inducing any significant changes in the circular dichroic spectra nor in the binding affinity for cyclomaltohexaose. Glu380, in cooperation with Glul86, therefore, is clearly indispensable for the liberation of p-maltose from starch. Substitutions of Leu383 by Ile and Gln, in contrast, led to remarkable increases in the K, values of both mutants when compared to that of the non-mutant enzyme. The mutants also showed marked reductions in their binding affinities to cyclomaltohexaose. Overall, it would appear that the k,,JK,,, of soybean pamylase increases in proportion to the length of the substrate molecule, and depends also on the characteristics of the side chain of the residue at position 383. Leu383, therefore, may be important for both substrate penetration and subsequent retention at the active site. Based on the foregoing, we propose an action mechanism of soybean P-amylase involving the interactions of three essential amino acid residues (AsplOl, Glu186 and Glu380) in concert with Leu383, and assumed an indispensable role for AsplOl.

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Monte Carlo simulation of multiple attack mechanism of β-amylase-catalyzed reaction

Nakatani

1997

Biopolymers

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β‐Amylase (EC 3.2.1.2) produces maltose (dimer) from the nonreducing ends of α‐1,4 glucosidic bonds of substrates like maltooligosaccharides, amylose, and amylopectin. The enzyme releases several maltose molecules from a single enzyme‐substrate complex without dissociation by multiple or repetitive attack containing many branching reaction paths. The Monte Carlo method was applied to the simulation of the β‐amylase‐catalyzed reaction including the multiple attack mechanism. The simulation starts from a single enzyme molecule and a finite number of substrate molecules. The selection of the substrate by the enzyme and degree of multiple attack proceeds by random numbers produced from a computer. The simulation was carried out until the whole substrate and the intermediate molecules were consumed. The simulated data were compared with experimental data of sweet potato β‐amylase using heptamer, octamer, nanomer, and 11‐mer as substrates. The only adjustable parameter for odd‐numbered substrates was the probability of multiple attack, while an additional adjustable parameter (a correction factor due to low reactivity of tetramer) was needed for even‐numbered substrates. © 1997 John Wiley & Sons, Inc. Biopoly 42: 831–836, 1997

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Evaluation of subsite affinities of soybean .BETA.-amylase by product analysis.

Cited by 15 publications

References 2 publications

Automated docking of ?-(1?4)- and ?-(1?6)-linked glucosyl trisaccharides and maltopentaose into the soybean ?-amylase active site

Automated docking of ?-(1?4)- and ?-(1?6)-linked glucosyl trisaccharides and maltopentaose into the soybean ?-amylase active site

Functional Analysis of Glu380 and Leu383 of Soybean β‐Amylase

Monte Carlo simulation of multiple attack mechanism of β-amylase-catalyzed reaction

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