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KMID : 0364819910290040230
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Korean Journal of Microbiology
1991 Volume.29 No. 4 p.230 ~ p.237
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Purification and Characterization of Nonmitochondrial Citrate Synthase from Saccharomyces cerevisiae
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Cho, Nam Seok/조남석
Kim, Kwang Soo/Maeng, Pil Jae/김광수/맹필재
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Abstract
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1
Citrate synthase 1 (mitochondrial) and citrate synthase 2 (nonmitochondrial) were purified from Saccharomyces cerevisiae. The physical and enzymatic characteristics of citrate synthase 2 were analyzed in comparison with citrate synthase 1. Both isoenzymes were shown to be dimeric proteins of identical subunits, and the molecular weights of the subunits were estimated to be 48.3 kDa for citrate synthase 1 and 47.0 kDa for citrate synthase 2, respectively. The optimal pH value for enzyme activity was pH 7.5 for both isoenzymes. However, the optimal temperature for the activity was strikingly different; while the activity of citrate synthase I reached its peak at 65°C, that of citrate synthase 2 was maximal at 40°C. Citrate synthase 2 showed, much lower thermal and pH stability than citrate synthase 1. In addition, citrate synthase -2 was affected much more by the metal ions such as Zn=+, Mn" and Co´+ than citrate synthase 1. Among the several possible regulatory metabolites tested, ATP showed the strongest inhibitory effect on both enzymes. ADP and NADH were found to have greater effect on citrate synthase 2 than on citrate synthase 1. Kinetic analysis revealed that citrate synthase 2 has approximately 7- and 3.5-fold lower affinity to acetyl CoA and to oxaloacetate, respectively,
than citrate synthase 1.
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