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Journal of Biochemistry 2005 137(2):141-145; doi:10.1093/jb/mvi027
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© 2005 The Japanese Biochemical Society

BIOCHEMISTRY

Mutagenesis of the Active Site Lysine 221 of the Pyruvate Kinase from Bacillus stearothermophilus

Hiroshi Sakai1

Department of Food and Nutritional Sciences, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526

1 For correspondence: Phone: +81-54-264-5576, Fax: +81-54-264-5099, E-mail: sakaih{at}u-shizuoka-ken.ac.jp

Lysine 221 of the pyruvate kinase from Bacillus stearothermophilus was mutated to arginine, leucine, asparatic acid and cysteine. All the mutated enzymes were 104 to 105 times less active than the wild-type enzyme. The cysteine-free enzyme C9S/C268S, and the enzyme C9S/C268S/K221C, which possessed a unique sulfhydryl group at position 221, were prepared. The former had comparable activity to the wild-type enzyme and the latter was 104 times less active. These enzymes were denatured and renatured after aminoethylation. The C9S/C268S/K221C enzyme failed to regain its activity when renatured without aminoethylation; but when it was renatured after aminoethylation, it regained 4.5% of the activity of the C9S/C268S enzyme. This evidence suggests the importance of the Lys221 for the pyruvate kinase activity. The kinetic parameters of the S-aminoethylated C9S/C268S/K221C enzyme suggest that it has decreased affinity for phosphoenolpyruvate.


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