Chorismate Mutase from Streptomyces aureofaciens: a Heat-Stable Enzyme

Abstract

Chorismate mutase from Streptomyces aureofaciens was purified 12-fold. This enzyme preparation did not show any activity when tested for anthranilate synthetase, prephenate dehydrogenase, or prephenate dehydratase. The catalytic activity of chorismate mutase has a broad optimum between pH 7 and 8. The initial velocity data followed regular Michaelis-Menten kinetics with a K. of 5.3 x 10-I M, and the molecular weight of the enzyme was determined by sucrose gradient centrifugation to be 50,000. Heat inactivation of chorismate mutase, which occurs above temperatures of 60 C, is reversible. The enzyme activity can be restored even when chorismate mutase is treated at the temperature of a boiling-water bath for 15 min. Heat-denatured and renatured enzymes showed the same Michaelis constant and the same molecular weight as the native enzyme. L-Phenylalanine, L-tyrosine, L-tryptophan, and metabolites of the aromatic amino acid pathway were tested as potential modifiers of chorismate mutase activity. The activity of the enzyme was inhibited by none of these substances. Chorismate mutase of S. aureofaciens was not repressed in cells grown in minimal medium supplemented with L-phenylalanine, L-tyrosine, or L-tryptophan.

On the mechanism of the chorismate mutase reaction

“…(1973). 6 Determined at 30 °C; Górisch & Lingens (1974). " Determined at 37 °C; Koch et al (1970a). "…”

“…The reaction follows first-order kinetics with a rate constant of 1.24 X 10-5 s_1 at 30 °C and 2.6 X 10~5 s-1 at 37 °C (Andrews et al, 1973). From the specific activity of the most highly purified preparation of chorismate mutase (EC 5.4.99.5) from Streptomyces aureofaciens (Górisch & Lingens, 1974), a turnover number of 300 s_1 at 30 °C can be calculated. The enzyme consists of three to four similar subunits so that two or even four catalytic sites per enzyme molecule are possible.…”

Unravelling the Mechanism of Elastin-like Polypeptide-Enzyme Fusion Stabilization in Organic Solvents

“…These samples were incubated at 37 °C for 20 min. The organic solvent was evaporated at 10 to 15 °C as it has very little enzyme activity . After this period, 10 μL of 10 M HCl was added to each sample.…”

“…The purified ELP−I-Cm2 was tested using the reaction buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 500 μM chorismate) along with a positive control, Cm2, and a (VPGVG) 40 (N40) negative control sequence. For each condition, ELP−I-Cm2 (either purified by ITC or organic 53 After this period, 10 μL of 10 M HCl was added to each sample. After 5 min, 15 μL of 15 M NaOH was added to each sample.…”

Inhibition of chorismate mutase activity of chorismate mutase-prephenate dehydrogenase from Aerobacter aerogenes

“…Chorismate mutase exists as a monofunctional enzyme in many species of microorganisms and higher plants. Streptomyces aureofaciens possesses such a mutase (Górisch & Lingens, 1973, 1974Górisch, 1978). The occurrence of bifunctional enzymes with chorismate mutase activity is common.…”