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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.

Purification procedures for phosphoenolpyruvate carboxylase from B. flavum were improved by using hydrophobic chromatography. The carboxylase showed optimum pH values of 7.2 and 8.0 with Mn2+ and Mg2+ as metallic activators, respectively. Purified phosphoenolpyruvate carboxylase was found to be synergistically inhibited by aspartate and 2-oxoglutarate in the absence or presence of an activator, acetyl-CoA. Similarly to the aspartate inhibition, 2-oxoglutarate alone inhibited the enzyme competitively with respect to both substrates, with an inhibitor constant of 4.7 mM. The dissociation constant for the combination of enzyme-2-oxoglutarate (-aspartate) complex with aspartate (2-oxoglutarate) was found to be one-third of that for the combination of the enzyme with aspartate (2-oxoglutarate). The Hill coefficient for phosphoenolpyruvate was increased from 1.0 to 2.3 by the simultaneous addition of the two inhibitors in a certain concentration range of phosphoenolpyruvate where strong synergistic effects were observed. Outside this concentration range, the coefficient was not altered or was slightly increased by the addition of aspartate, 2-oxoglutarate, or both. The synergistic action seems to be caused by these effects, in addition to the decrease in dissociation constants of the inhibitors. Hill coefficients for aspartate and 2-oxoglutarate were both approximately 2.0. The coefficient for one inhibitor did not vary with the addition of the other inhibitor. Although many structural analogues of the two inhibitors, such as 2-oxoadipate and 3-hydroxyaspartate, were very weak inhibitors, their synergistic effects with aspartate or 2-oxoglutarate were comparable to the effects of the two natural inhibitors. On the other hand, malate and succinate, which markedly inhibited the enzyme, did not show synergistic action with aspartate or 2-oxoglutarate. Hill coefficients for the structural analogues showing synergistic effects were approximately 2.0 or above, whereas those for malate and succinate, which did not enhance the inhibitions, were about 1.0. Phosphoenolpyruvate carboxylase from an aspartate-producing mutant had the inhibitor constant of 5.8 mM for 2-oxoglutarate, i.e., slightly higher than wild-type enzyme. The inhibitor constant for aspartate was three times higher than that of the wild-type enzyme as reported previously. The dissociation constant for aspartate of the enzyme-aspartate-2-oxoglutarate complex in the mutant enzyme was 8 times that in the wild-type enzyme, indicating that weaker synergistic inhibition was observed with the mutant enzyme.

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A Brevibacteriumflavum mutant lacking pyruvate kinase, No. 70, grew on glucose, fructose and sucrose as well as the original wild strain did, but was unable to grow on ribose or gluconate unless pyruvate was added. Mutants that required pyruvate for growth on ribose were derived directly from the wild strain. Manyof them were completely or partially defective in pyruvate kinase activity. These pyruvate kinase mutants were also unable to grow on gluconate. A phosphoenolpy-ruvate (PEP) : sugar phosphotransferase system (PTS) was found in B. flavum, which catalyzed the formation ofpyruvate and sugar phosphate from PEPand sugar. The system required Mg2+ , acted on glucose, fructose, mannose, glucosamine and 2-deoxyglucose, and existed in the cells grown on any of the carbon sources tested. Cells grown on fructose, mannitol and sucrose, however, exhibited higher PTS activities on fructose than those grown on others. Glucose PTSactivity was about 20-fold stronger than that of glucokinase. Other sugar metabolic enzymes, inducible mannitol dehydrogenase, gluconokinase, ribokinase and maltase, as well as constitutive invertase were also detected. Oxaloacetate decarboxylase and malic enzyme, which also catalyzed the pyruvate formation, were found in B. flavum, but the latter activity was very low in cells grown on glucose. The levels of these enzymesin pyruvate kinase mutants unable to grow on ribose or gluconate derived from the wild strain were almost identical to those in the wild-type strain. Since phosphoenolpyruvate (PEP) is a branch-point intermediate in the sugar metabolism for amino acid biosynthesis, studies on enzymes catalyzing PEP metabolism seem interesting, especially for an amino acid-producing bacterium, Brevibacterium flavum. Studies on PEP carboxylase1} which catalyzes the formation of oxaloacetate, an intermediate in amino acid biosynthesis, and on pyruvate kinase (PK)2) which catalyzes the first step of degradation of PEP to CO2 were reported previously. Pyruvate produced from PEP via the PK reaction is not only oxidized to yield CO2but also used for the biosynthesis of various essential cell constituents. The growth on glucose of mutants lacking PK, however, was substantially identical to that of the wild-type strain, suggesting that enzymesother than PKmight serve to form sufficient pyruvate for growth. The present study revealed that the PEP : sugar phosphotransferase system (PTS) as well as PK significantly contribute to the pyruvate formation during growth on glucose, whereas only PK is responsible for the formation during growth on sugars which are not metabolized via the PTS reaction. MATERIALS AND METHODS Chemicals. PEP, ADPand ATP were purchased from Sigma Chemical Co., dithiothreitol from Calbiochem, and NAD, NADHand NADPfrom Boehringer Mannheim GmbH.All the enzymes used for enzyme assays and for the metabolite determinations were obtained from the latter company. Abbreviations: PK, pyruvate kinase; PTS, phosphotransferase system; LDH, lactate dehydrogenase; PEP, phosphoenolpyruvate; FBP, fr...

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