TY - JOUR
T1 - Metabolic studies on citrate synthase mutants of yeast. A change in phenotype following transformation with an inactive enzyme
AU - Kispal, G.
AU - Evans, C. T.
AU - Malloy, C.
AU - Srere, P. A.
N1 - Copyright:
Copyright 2004 Elsevier B.V., All rights reserved.
PY - 1989
Y1 - 1989
N2 - We have studied the growth on acetate, the metabolism of acetate enzymes, and respiration of a series of citrate synthase mutants of Saccharomyces cerevisiae. The results confirmed and extended our previous observation that cytosolic citrate synthase is not necessary for growth on acetate. Deletion of mitochondrial citrate synthase (CS1) protein resulted in changes in metabolites, decrease in the amounts of pyruvate and α-ketoglutarate dehydrogenase complexes, reduced mitochondrial respiration of citrate and isocitrate, and an inability to grow on acetate. Using site-directed mutagenesis, we constructed two separate CS1 proteins with mutations in the enzyme's active site. The mitochondria of cells carrying either site-directed mutagenized CS1 contained the inactive citrate synthase protein. With one mutant in which His313 was replaced with a glycine (CS1/H313G), growth on acetate was restored, and mitochondrial respiration of citrate and isocitrate increased toward parental levels as did the levels of several enzymes. With the other mutant CS1 in which Asp414 was replaced with a glycine (CS1/D414G), no growth on acetate or changes in other parameters was observed. We propose that the characteristics of the strain carrying the CS1 with a H313G mutation result from the formation of an intact Krebs cycle complex by the inactive but structurally unchanged H313G protein.
AB - We have studied the growth on acetate, the metabolism of acetate enzymes, and respiration of a series of citrate synthase mutants of Saccharomyces cerevisiae. The results confirmed and extended our previous observation that cytosolic citrate synthase is not necessary for growth on acetate. Deletion of mitochondrial citrate synthase (CS1) protein resulted in changes in metabolites, decrease in the amounts of pyruvate and α-ketoglutarate dehydrogenase complexes, reduced mitochondrial respiration of citrate and isocitrate, and an inability to grow on acetate. Using site-directed mutagenesis, we constructed two separate CS1 proteins with mutations in the enzyme's active site. The mitochondria of cells carrying either site-directed mutagenized CS1 contained the inactive citrate synthase protein. With one mutant in which His313 was replaced with a glycine (CS1/H313G), growth on acetate was restored, and mitochondrial respiration of citrate and isocitrate increased toward parental levels as did the levels of several enzymes. With the other mutant CS1 in which Asp414 was replaced with a glycine (CS1/D414G), no growth on acetate or changes in other parameters was observed. We propose that the characteristics of the strain carrying the CS1 with a H313G mutation result from the formation of an intact Krebs cycle complex by the inactive but structurally unchanged H313G protein.
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M3 - Article
C2 - 2661555
AN - SCOPUS:0024319155
SN - 0021-9258
VL - 264
SP - 11204
EP - 11210
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 19
ER -