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Báo cáo khoa học: Effects of replacing active site residues in a cold-active alkaline phosphatase with those found in its mesophilic counterpart from Escherichia coli

Alkaline phosphatase (AP) from a North Atlantic marineVibriobacterium was previously characterized as being kinetically cold-adapted. It is still unknown whether its characteristics originate locally in the active site or are linked to more general structural factors. | ỊFEBS Journal Effects of replacing active site residues in a cold-active alkaline phosphatase with those found in its mesophilic counterpart from Escherichia coli Katrin Gudjonsdottir and Bjarni Asgeirsson Department of Biochemistry Science Institute University of Iceland Reykjavik Iceland Keywords cold adaptation metalloenzyme mutagenesis protein stability psychrophilic Correspondence B. Asgeirsson Science Institute University of Iceland Dunhaga 3 107 Reykjavik Iceland Fax 354 552 89 11 Tel 354 525 48 00 E-mail bjarni@raunvis.hi.is Received 26 September 2007 revised 3 November 2007 accepted 7 November 2007 doi 10.1111 j.1742-4658.2007.06182.x Alkaline phosphatase AP from a North Atlantic marine Vibrio bacterium was previously characterized as being kinetically cold-adapted. It is still unknown whether its characteristics originate locally in the active site or are linked to more general structural factors. There are three metal-binding sites in the active site of APs and all three metal ions participate in catalysis. The amino acid residues that bind the two zinc ions most commonly present are conserved in all known APs. In contrast two of the residues that bind the third metal ion numbered 153 and 328 in Escherichia coli AP are different in various APs. This may explain their different catalytic efficiencies as the Mg2 most often present there is important for both structural stability and the reaction mechanism. We have mutated these key residues to the corresponding residues in E. coli AP to obtain the double mutant Asp116 Lys274 and both single mutants. All these mutants displayed reduced substrate affinity and lower overall reaction rates. The Lys274 and Asp116 Lys2.74 mutants also displayed an increase in global heat stability which may be due to the formation of a stabilizing salt bridge. Overall the results show that a single amino acid substitution in the active site is sufficient to alter the structural stability of the cold-active Vibrio AP both locally