Metabolic studies on citrate synthase mutants of yeast. A change in phenotype following transformation with an inactive enzyme

G. Kispal, C. T. Evans, C. Malloy, P. A. Srere

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)11204-11210
Number of pages7
JournalJournal of Biological Chemistry
Volume264
Issue number19
StatePublished - 1989

Fingerprint

Citrate (si)-Synthase
Yeast
Acetates
Yeasts
Phenotype
Enzymes
Respiration
Growth
Citric Acid
Glycine
Proteins
Ketoglutarate Dehydrogenase Complex
Pyruvate Dehydrogenase Complex
Mutagenesis
Mutation
Mitochondria
Citric Acid Cycle
Metabolites
Site-Directed Mutagenesis
Pyruvic Acid

ASJC Scopus subject areas

  • Biochemistry

Cite this

Metabolic studies on citrate synthase mutants of yeast. A change in phenotype following transformation with an inactive enzyme. / Kispal, G.; Evans, C. T.; Malloy, C.; Srere, P. A.

In: Journal of Biological Chemistry, Vol. 264, No. 19, 1989, p. 11204-11210.

Research output: Contribution to journalArticle

@article{57ec209fd3d64649b224202492e9a10f,
title = "Metabolic studies on citrate synthase mutants of yeast. A change in phenotype following transformation with an inactive enzyme",
abstract = "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.",
author = "G. Kispal and Evans, {C. T.} and C. Malloy and Srere, {P. A.}",
year = "1989",
language = "English (US)",
volume = "264",
pages = "11204--11210",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "19",

}

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.

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.

UR - http://www.scopus.com/inward/record.url?scp=0024319155&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024319155&partnerID=8YFLogxK

M3 - Article

VL - 264

SP - 11204

EP - 11210

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 19

ER -