Engineering the properties of a cold active enzyme through rational redesign of the active site

In an effort to explore the effects of local flexibility on the cold adapta tion of enzymes, we designed point mutations aiming to modify side-chain fl exibility at the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutagenesis targets were residues Trp260 an d Ala219 of the catalytic site and His135 of the Mg2+ binding site. The rep lacement of Trp260 by Lys in mutant W260K, resulted in an enzyme less activ e than the wild-type in the temperature range 5-25 degreesC. The additional replacement of Ala219 by Asn in the double mutant W260K/A219N, resulted in a drastic increase in the energy of activation, which was reflected in a c onsiderably decreased activity at temperatures of 5-15 degreesC and a signi ficantly increased activity at 20-25 degreesC. Further substitution of His1 35 by Asp in the triple mutant W260K/A219N/H135D restored a low energy of a ctivation. In addition, the His135-->Asp replacement in mutants H135D and W 260K/A219N/ H135D resulted in considerable stabilization. These results sug gest that the psychrophilic character of mutants can be established or mask ed by very slight variations of the wildtype sequence, which may affect act ive site flexibility through changes in various conformational constraints.