No abstract

Brevibacteriumflavum mutants defective in the phosphoenolpyruvate (PEP)-dependent glucose phosphotransferase system (PTS) were selected with high frequency by 2-deoxyglucose-resistance. Most of them (DOGr) still had the fructose-PTS and grew not only on fructose but also on glucose like the wild-type strain. A mutant having l/8th the fructose-PTS activity of the wild strain but normal glucose-PTS activity was isolated as a xylitol-resistant mutant. It grew on glucose but not on fructose. The glucose-PTS was active on glucose, glucosamine, 2-deoxyglucose and mannose, and slightly on methyl-a-glucoside and TV-acetylglucosamine, but not on fructose or xylitol. The fructose-PTS acted on fructose and xylitol, and to someextent on glucose but not on glucosamine or 2-deoxyglucose. Mutants unable to grow on glucose (DOGrGlc~)derived from a DOGrmutant were all defective in the fructose-PTS. All revertants able to grow on glucose derived from a DOGrGlc~mutant had the fructose-PTS. The glucokinase activity was about 2/3rds the glucose activity of the fructose-PTS. All the DOGrGlc"mutants had normal levels of glucokinase. One of these mutants grew on maltose and sucrose, which were hydrolyzed to glucose. Thus, glucokinase seems to contribute to the phosphorylation of glucose liberated inside the cell. The fructose-PTS was induced by fructose and repressed by glucose. The glucose repression was not observed in a mutant defective in the glucose-PTS.In Brevibacteriumflavum, an industrial bacterium producing amino acids, glucose is me- 3) The role of glucokinase in glucose metabolism was not revealed. If glucose is phosphorylated solely at the expense of PEP, the theoretical yield in the fermentative production of amino acids synthesized from PEP will be greatly affected. The present paper deals with the derivation of PTS mutants, properties of the individual PTSs and the roles of the PTSs and glucokinase in sugar metabolism.

Phosphoenolpyruvate (PEP) carboxylases (PC) were purified from a wild strain and an aspartate-producing mutant of Brevibacterium flavum to electrophoretic homogeneity. The molecular weights of the enzymes were determined to be 4.1 X 10(5) by the gel-filtration technique. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme gave only one protein band with a molecular weight of 1.07 X 10(5). The enzyme was labile and stabilized by substrate PEP, activators, metallic cofactors, an allosteric inhibitor and ammonium sulfate. The mechanism for the PC reaction was rapid equilibrium random Bi Bi with a dead end complex, enzyme-bicarbonate-Pi. The KmS for PEP and bicarbonate were 2.5 and 0.63 mM, respectively, and the apparent KmS were not affected by the secondary substrate concentrations. Dissociation constants for Pi of enzyme-Pi and the dead end complex were 5.0 and 16 mM, respectively. Aspartate inhibition was completely competitive with both the substrates, PEP and bicarbonate, with an inhibitor constant of 0.044 mM. An activator, acetyl-CoA, did not alter the apparent Km for bicarbonate but decreased that for PEP. The activator constants for the enzyme-PEP complex and free enzyme were 6.3 and 40 microM, respectively. Double reciprocal plots of reaction rate against PEP concentration were not linear at lower PEP concentrations. Hill coefficients for PEP were 1.6 in the absence of any effectors, 1.0 in the presence of acetyl-CoA, and 2.3 in the presence of aspartate. As to the mutant enzyme, only the inhibitor constant for aspartate was increased, being 0.18 mM, but other constants, coefficients, as described above, and specific activity were almost the same as those of the wild-type enzyme.