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Title 

Mutagenesis of Ala290, which modulates substrate subsite affinity at the catalytic interface of dimeric ThMA

Authors 

S H ParkH ChaH K KangJ H ShimEui-Jeon WooJ W KimK H Park

Publisher 

Elsevier

Issue Date 

2005

Citation 

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, vol. 1751, no. 2, pp. 170-177

Keywords 

kinetic parametermaltogenic amylase (ThMA)maltosethermus sp.enzyme substrate complexsite directed mutagenesiscatalytic domainmutagenesis, site-directedsubstrate specificity

Abstract 

The goal of this study was to develop a maltose-producing enzyme using protein engineering and to clarify the relation between the substrate specificity and the structure of the substrate-binding site of dimeric maltogenic amylase isolated from Thermus (ThMA). Ala290 at the interface of ThMA dimer in the vicinity of the substrate-binding site was substituted with isoleucine, which may cause a structural change due to its bulky side chain. TLC analysis of the action pattern of the mutant ThMA-A290I, using maltooligosaccharides as substrates, revealed that ThMA-A290I used maltotetraose to produce mostly maltose, while wild-type ThMA produced glucose as well as maltose. The wild-type enzyme eventually hydrolyzed the maltose produced from maltotetraose into glucose, but the mutant enzyme did not. For both enzymes, the cleavage frequency of the glycosidic bond of maltooligosaccharides was the highest at the second bond from the reducing end. The mutant ThMA had a much higher Km value for maltose than the wild-type ThMA. The kinetic parameter, kcat/Km, of ThMA-A290I for maltose was 48 times less than that of wild-type ThMA, suggesting that the subsite affinity and hydrolysis mode of ThMA were modulated by the residue located at the interface of ThMA dimer near the active site. The conformational rearrangement in the catalytic interface probably led to the change in the substrate binding affinity of the mutant ThMA. Our results provide basic information for the enzymatic preparation of high-maltose syrup.

ISSN 

1570-9639

Link 

http://dx.doi.org/10.1016/j.bbapap.2005.05.004

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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