| Summary: | In order to explore the effects of local flexibility on the cold adaptation of enzymes, we designed point mutations aiming to modify side chain flexibility at the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutations were designed based on multiple sequence alignment, assisted by a homology based model. The mutagenesis targets were residues Trp-260 and Ala-219 of the catalytic site and His-135 of the Mg2+ binding site. The replacement of Trp-260 by Lys in mutant W260K, resulted in a less active than the wild-type enzyme in the temperature range 5-25oC. The additional replacement of Ala-219 by Asn in the double mutant W260K/A219N, resulted in a drastic increase in the energy of activation which is reflected in a considerably decreased activity at temperatures 5-15oC and a significantly increased activity at 20-25oC. Further substitution of His135 by Asp in the triple mutant W260K/A219N/H135D restored the low energy of activation. In addition, the His135 to Asp replacement in mutants H135D and w260K/A219N/H135D resulted in a considerable stabilization. Furthermore, we designed point mutations aiming to alter the distribution of glycine residues close to the active site of the psychrophilic alkaline phosphatase. The mutagenesis targets were residues Gly-261 and Gly-262. The replacement of Gly-262 by Ala resulted in an inactive enzyme. Substitution of Gly-261 by Ala resulted in an enzyme with lower stability and increased energy of activation. The double mutant G261A/Y269A designed on the basis of side-chain packing criteria from a modeled structure of the enzyme resulted in restoration of the energy of activation to the levels of the native enzyme and in an increased stability compared to the mutant G261A. It seems therefore, that the Gly cluster in combination with its structural environment plays a significant role in the cold adaptation of the enzyme. These results suggest that the psychrophilic character of mutants can be established or masked by very slight ...
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