Two cysteine residues were introduced to selected positions (Serine-309 and Serine-43) of the Thermoplasma acidophilum citrate synthase. Serine-309 Cysteine mutation drastically reduced the activity, as well as the thermostabiity of the enzyme. | Tr. J. of Biology 23 (1999) 423–431 © TÜBİTAK Purification and Characterization of Cysteine-43 Mutant Citrate Synthase from Thermoplasma acidophilum İpek ERDURAN Department of Biology, Middle East Technical University, 06531, Ankara-TURKEY Semra KOCABIYIK* Department of Biology, Middle East Technical University, 06531, Ankara-TURKEY Received: Abstract: Two cysteine residues were introduced to selected positions (Serine-309 and Serine-43) of the Thermoplasma acidophilum citrate synthase. Serine-309 Cysteine mutation drastically reduced the activity, as well as the thermostabiity of the enzyme. Although Serine-43 Cysteine mutation reduced the enzyme activity, it did not have any effect on the thermostability. Both wild-type and cysteine-43 mutant Thermoplasma citrate synthase enzymes were purified to homogenity using affinity chromatography on Matrex Gel Red A. The cysteine-43 mutant Thermoplasma citrate synthase was strikingy similar to the wild-type recombinant citrate synthase in its molecular size, substrate and cofactor specificities, pH profile and thermal resistance. The decrease in the specific activity of the citrate synthase as a result of this mutation was reflected by V max and E a values. Key Words: Thermostability, Thermoplasma acidophium, citrate synthase, site-specific mutagenesis. Thermoplasma acidophium Orijinli Sistin-43 Mutant Sitrate Sentaz Enziminin Saflaştırılması ve Karakterizasyonu Özet: Thermoplasma acidophium sitrat sentaz enziminin seçilmiş iki pozisyonuna (ser-309 ve Ser-43) iki sistin grubu yerliştirimiştir. Serin 309 Sistin mutasyonu enzimin termostabilitesi kadar aktivitesini de büyük ölçüde azatmıştır. Serin 43 Sistin mutasyonu her ne kadar enzimin aktivitesini azaltmışsa da termostabiliteyi etkilememiştir. Hem mutant olmayan, hem de Sistin-43 mutant Thermoplasma sitrat sentaz enzimeri Matrex Gel Red A kullanılarak yapılan afinite kromatografi ile homojen bir şekilde safaştırılmıştır. Sistin-43 mutant Thermoplasma .
