Sep 25, 1976

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase. Purification, properties, and kinetics of the tyrosine-sensitive isoenzyme from Escherichia coli

The Journal of Biological Chemistry
R Schoner, K M Herrmann

Abstract

The tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (7-phospho-2-keto-3-deoxy-D-arabino-heptonate D-erythrose-4-phosphate lyase (pyruvate-phosphorylating), EC 4.2.1.15) was purified to homogeneity from extracts of Escherichia coli K12. A spectrophotometric assay of the enzyme activity, based on the absorption difference of substrates and products at 232 nm, was developed. The enzyme has a molecular weight of 66,000 as judged by gel filtration on Sephadex G-200, and a subunit molecular weight of 39,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. This suggests either a rapid monomer-dimer equilibrium, or a very asymmetric shape for the native enzyme. The enzyme shows a narrow pH optimum around pH 7.0. The enzyme is stable for several months when stored at -20 degrees in phosphate buffer containing phosphoenol-pyruvate. Intersecting lines in double reciprocal plots of initial velocity data at substrate concentrations in the micromolar range suggest a sequential mechanism with-catalyzed reaction. Product inhibition studies specify an ordered sequential BiBi mechanism with a dead-end E-P complex. The feedback inhibitor tyrosine at concentrations above 10 muM ...Continue Reading

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Mentioned in this Paper

Alkalescens-Dispar Group
Aldehyde-Lyases
2-Dehydro-3-Deoxyphosphoheptonate Aldolase
Plasma Protein Binding Capacity
Drug Stability
Hydrogen-Ion Concentration
Mathematics

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