Studies on cell-free metabolism: Ethanol production by a yeast glycolytic system reconstituted from purified enzymes

Abstract A reconstituted glycolytic system has been established from individually purified enzymes to simulate the conversion of glucose to ethanol plus CO 2 by yeast. Sustained and extensive conversion occurred provided that input of glucose matched the rate of ATP degradation appropriately. ATPase activity could be replaced by arsenate, which uncoupled ATP synthesis from glycolysis. The mode of uncoupling was investigated, and it was concluded that the artificial intermediate, 1-arseno-3-phosphoglycerate, has a half-life of no more than a few milliseconds. Arsenate at 4 mM concentration could simulate the equivalent of 10 μmol ml −1 min −1 of ATPase activity. The reconstituted enzyme system was capable of totally degrading 1 M (18% w/v) glucose in 8 h giving 9% (w/v) ethanol. The levels of metabolites during metabolism were measured to detect rate-limiting steps. The successful operation of the reconstituted enzyme system demonstrates that it is possible to carry out complex chemical transformations with multiple enzyme systems in vitro.

[1]  K. Pye,et al.  Sustained sinusoidal oscillations of reduced pyridine nucleotide in a cell-free extract of Saccharomyces carlsbergensis. , 1966, Proceedings of the National Academy of Sciences of the United States of America.

[2]  S. Yun,et al.  A revised preparation of yeast (Saccharomyces cerevisiae) pyruvate kinase. , 1976, The Journal of biological chemistry.

[3]  R. Scopes,et al.  Inhibition of muscle phosphorylase a by natural components of the sarcoplasm. , 1981, Archives of biochemistry and biophysics.

[4]  K. Uğurbil,et al.  Phosphorus-31 nuclear magnetic resonance studies of the effect of oxygen upon glycolysis in yeast. , 1981, Biochemistry.

[5]  W. Rutter,et al.  [87] Fructose diphosphate aldolase , 1966 .

[6]  D. Needham,et al.  The coupling of oxido-reductions and dismutations with esterification of phosphate in muscle. , 1937, The Biochemical journal.

[7]  L. Byers,et al.  Interaction of phosphate analogs with glyceraldehyde-3-phosphate dehydrogenase , 1979 .

[8]  R. Shulman,et al.  Phosphorus-31 nuclear magnetic resonance studies of wild-type and glycolytic pathway mutants of Saccharomyces cerevisiae. , 1979, Biochemistry.

[9]  I. Kulaev,et al.  Vacuoles: main compartments of potassium, magnesium, and phosphate ions in Saccharomyces carlsbergenis cells , 1980, Journal of bacteriology.

[10]  R. Scopes.  An improved procedure for the isolation of 3-phosphoglycerate kinase from yeast. , 1971, The Biochemical journal.

[11]  R K Scopes,et al.  Purification of 3-phosphoglycerate kinase from diverse sources by affinity elution chromatography. , 1978, The Biochemical journal.

[12]  R. Scopes.  Automated fluorometric analysis of biological compounds. , 1972, Analytical biochemistry.

[13]  E. Kendall Pye,et al.  Biochemical mechanisms underlying the metabolic oscillations in yeast , 1969 .

[14]  M. Ciriacy,et al.  Physiological Effects of Seven Different Blocks in Glycolysis in Saccharomyces cerevisiae , 1979, Journal of bacteriology.

[15]  J. Gancedo,et al.  Activation by phosphate of yeast phosphofructokinase. , 1977, The Journal of biological chemistry.

[16]  J. Gancedo,et al.  Concentrations of intermediary metabolites in yeast. , 1973, Biochimie.

[17]  R. Scopes,et al.  Control of substrate cycling at fructose phosphates in a reconstituted muscle glycolytic system. , 1981, Archives of biochemistry and biophysics.

[18]  R K Scopes,et al.  Measurement of protein by spectrophotometry at 205 nm. , 1974, Analytical biochemistry.

[19]  R. Gillies,et al.  31P NMR studies of intracellular pH and phosphate metabolism during cell division cycle of Saccharomyces cerevisiae. , 1981, Proceedings of the National Academy of Sciences of the United States of America.