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Mutagenesis of Ala290, which modulates substrate subsite affinity at the catalytic interface of dimeric ThMA : Thermus maltogenic amylase의 기질 결합친화성을 조절하는 Ala290 잔기의 역할

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Authors

박성훈

Advisor
박관화
Issue Date
2005
Publisher
서울대학교 대학원
Keywords
Kinetic parametersKinetic parametersmaltogenic amylase from Thermus sp. (ThMA)maltogenic amylase from Thermus sp. (ThMA)기질친화성maltose producing enzymecleavage frequencysubsite affinitydimeric ThMAcleavage frequency말토스 생산 효소dimeric ThMA.
Description
Thesis(master`s)--서울대학교 대학원 :농생명공학부,2005.
Abstract
The goal of this study was to develop a maltose-producing enzyme using protein engineering and
to clarify the relationship between the substrate specificity and the structure of the
substrate-binding site of dimeric maltogenic amylase isolated from Thermus (ThMA). The four
amino acids residues (G50, D109, A290, and V431) at the interface between ThMA subunits were
substituted with bulky residues like isoleucine and glutamic acid to induce structural change of
substrate binding site. G50I/D109E and G50I/D109E/V431I resulted in less hydrolyzing activity
for amylopectin compared to amylose, thereby enhancing the discriminating ability between small
and large size substrates. The replacements with the bulky amino acid residues changed the
geometry of the catalytic site groove so that large molecule could not get in contact with the
active site.
Interestingly, TLC action pattern analysis of the mutant A290I with maltooligosaccharides
revealed that mutant enzyme produced mostly maltose from maltotetraose, whereas wild-type enzyme
produced glucose and 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 46 times less than that of the
wild-type ThMA, suggesting that the subsite affinity and hydrolysis mode of ThMA are modulated
by residues 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 maltogenic amylase ThMA. These results could provide basic information
for the enzymatic preparation of high-maltose syrup.
Language
English
URI
http://dcollection.snu.ac.kr:80/jsp/common/DcLoOrgPer.jsp?sItemId=000000051527

https://hdl.handle.net/10371/67561
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