Amino Acid Residues Specific for the Catalytic Action towards α-1,6-Glucosidic Linkages in Klebsiella Pullulanase
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概要
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Mutantios were introduced at residues His607,Asp677,His682,and His833 in pullulanase from Klebsiella aerogenes in order to probe the role of these amino acdi residues, which are located in the four conserved regions of the α-amylase family, in the action of the enzyme towards α-1,6-glucosidic linkages. For the mutations, His was replaced by Asn and Ala, and Asp by Asn and Ser. Amino acdi substitutions for His607,Asp677,or His833 resulted in complete loss of enzyme activity. In contrast, the mutations at His682 still retained their activities. The binding affinity of these variants for α- or β-cyclodextrine (CD), which are competitive inhibitors for pululanase, was measured using an α-CD Sepharose column. The mutations at His833 did not change the ninding affinity for α-CD, whereas the mutations at His607 or Asp677 resulted in these two variants losing their binding ability towards pullulan. These results suggeste that in Klevsiella pullulanase, His607and Asp677 perticipate in substrate binding and His833 is involved in catalysis, but His682 may be not in active site. We also found new amino acdi consensus sequences specific for starch debranching enzymes in two oligo-1,6-glucosidases, several pullulanases, and an isoamylase. Two amino acid residues in the predicited consensus region of Klebsiella pullulanase, Tyr559 and Tyr564,were replaced by Ala or Phe. The Tyr559 variants resulted in complete loss of pullulanase activity without seriously affecting the binding affinities for α-CD and pullulan. The mutations at Tyr564 did nor completely inactivate the enzymes, but dramatically decreased the activity. Thus, the region in Klebsiella pullulanase that includes Tyr559-Tyr564 probably participates in catalysis specific towards α-1,6-glucosidic linkages in starch debranching enzymes.
- 公益社団法人日本生物工学会の論文
- 1997-10-25
著者
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Yamashita Mitsuo
Department Of Biotechnology Graduate School Of Engineering Osaka University:department Of Applied Ch
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Murooka Yoshikatsu
Depart. Of Bioteclnology Graduate School Of Eng. Osaka Univ.
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Yamashita Mitsuo
Department Of Agricultural Chemistry Faculty Of Bioresources Mie University Department Of Biotechnol
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MATSUMOTO DAI
Department of Biotechnology, Graduate School of Engineering, Osaka University
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Matsumoto Dai
Department Of Biotechnology Graduate School Of Engineering Osaka University
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Murooka Yoshikatsu
Department of Biotechnology, Graduate School of Engineering, Osaka University
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