Characterization of 5-Enolpyruvylshikimate-3- phosphate Synthase from Sclerotinia sclerotiorum / 中国生物化学与分子生物学报

ABSTRACT

The 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase activity of Sclerotinia sclerotiorum is one of the multifunctional enzyme AROM activities, which catalyzes a reversible conversion of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to EPSP and inorganic phosphate, and is inhibited by the herbicide glyphosate (N-phosphonomethyl glycine). AROM protein has been purified from Sclerotinia sclerotiorum and the EPSP synthase has been analyzed. The results indicated that the optimal pH and temperature of EPSP synthase were 7.2 and 30℃ respectively. The activation energy of the heat-deactivated reaction of the enzyme was found to be 69.62 kJ/mol. Both of the substrates, S3P and PEP, were showed to inhibit the reaction rate when their concentrations exceeded 1 mmol/L and 2 mmol/L respectively. The Km of 140.98 μmol/L for PEP and 139.58 μmol/L for S3P were obtained by Dalziel equation which was a steadystate kinetic equation of the enzymatic reaction with the double substrates. The kinetic pattern of the enzyme was consistent with a sequential mechanism. Inhibition of the EPSP synthase reaction by glyphosate was competitive with respect to PEP, with the Ki 0. 32 μmol/L, and noncompetitive with regard to S3P. Activation by [ K+ ] was observed in the forward reaction. The Km (PEP) was lowered by increasing [ K+ ], while the Km (S3P) changed irregularly and the Ki (PEP) was enhanced.