Effect of Enzyme Concentration on Regulation of Dissociable Chorismate Mutase in Brevibacterium flavum

• Published in: Journal of biochemistry (Tokyo) 1981-01, Vol.89 (5), p.1483-1492
• Main Authors: SHIIO, Isamu, SUGIMOTO, Shin-ichi
• Format: Article
• Language: English
• Subjects: 3-Deoxy-7-Phosphoheptulonate Synthase - metabolism; Brevibacterium - enzymology; Brevibacterium flavum; Chorismate Mutase - antagonists & inhibitors; Chorismate Mutase - metabolism; Feedback; Isomerases - metabolism; Kinetics; Molecular Weight; Phenylalanine - pharmacology; Polyethylene Glycols - pharmacology; Potassium Chloride - pharmacology; Tryptophan - pharmacology; Tyrosine - pharmacology
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a133341

Deviation from linear relationship between enzyme concentration and activity in chorismate mutase of Brevibacterium flavum, the first enzyme specific for phenyl-alanine and tyrosine biosynthesis, increased and decreased upon the addition of KC1 and polyethyleneglycol, respectively. Furthermore, deviation decreased as substrate concentration increased. Gel-chromatographical experiments showed that the deviation from linearity was due to dissociation of chorismate mutase into two inactive components on dilution of the enzyme. The dissociation constant was calculated to be 1.1 × 10−4 (μM)4 under the standard assay conditions. Phenylalanine and tyrosine, feedback inhibitors, increased the deviation from linearity, and dissociation constant of the enzyme 35- and 9-fold, respectively. Tryptophan, the activator, decreased the deviation and the constant to one-twenty eight. Phenylalanine, tyrosine and tryptophan showed 44 and 35% inhibitions, and 2-fold activation, respectively, at a high enzyme concentration where the enzyme did not dissociate even in the presence of the effectors. These effects were much weaker than those at a low concentration, which involved indirect effects by these on dissociation. Intracellular concentration of the enzyme of the wild strain corresponded to a high region of enzyme concentration as above. Concentrations of phenylalanine and tyrosine giving 50% inhibition of the high concentration of chorismate mutase in the presence and absence of 0.01 mM tryptophan were 750 and 50 μM, respectively. These concentrations were similar to or much higher than that for 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthetase, the first enzyme in the common pathway. An enzymatic model for examining the effects of feedback control of chorismate mutase and DAHP synthetase on steady state concentration of chorismate under physiological conditions showed that an increase in phenylalanine and tyrosine concentrations resulted in a decrease in chorismate concentration. It was concluded that synthesis of phenylalanine and tyrosine was regulated by the control of DAHP synthetase under physiological conditions, in contrast to the previous suggestion that it was regulated by control of chorismate mutase. Accumulation of chorismate in culture broth suggested from previous results was not detected in the wild strain, but was detected only when a mutant lacking chorismate mutase was cultured under the tyrosine-limiting conditions where the synergistic feedb