AMINO-ACID-RESIDUES SPECIFIC FOR THE CATALYTIC ACTION TOWARDS ALPHA-1,6-GLUCOSIDIC LINKAGES IN KLEBSIELLA PULLULANASE

Mutations mere introduced at residues His607, Asp677, His682, and His8 33 in pullulanase from Klebsiella aerogenes in order to probe the role of these amino acid residues, which are located in the four conserved regions of the alpha-amylase family, in the action of the enzyme towa rds alpha-1,6-glucosidic linkages. For the mutations, His was replaced by Asn and Ala, and Asp by Asn and Ser. Amino acid 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 alpha- or beta-cyclodextr ine (CD), which are competitive inhibitors for pullulanase, was measur ed using an alpha-CD Sepharose column. The mutations at His833 did not change the binding affinity for alpha-CD, whereas the mutations at Hi s607 or Asp677 resulted in these two variants losing their binding abi lity towards pullulan. These results suggest that in Klebsiella pullul anase, His607 and Asp677 participate in substrate binding and His833 i s involved in catalysis, but His682 may be not in the active site. We also found new amino acid consensus sequences specific for starch debr anching enzymes in two oligo-1,6-glucosidases, several pullulanases, a nd an isoamylase. Two amino acid residues in the predicted consensus r egion of Klebsiella pullulanase, Tyr559 and Tyr564, were replaced by A la or Phe. The Tyr559 variants resulted in complete loss of pullulanas e activity without seriously affecting the binding affinities for alph a-CD and pullulan. The mutations at Tyr564 did not completely inactiva te the enzymes, but dramatically decreased the activity. Thus, the reg ion in Klebsiella pullulanase that includes Tyr559-Tyr564 probably par ticipates in catalysis specific towards alpha-1,6-glucosidic linkages in starch debranching enzymes.