PHOTOOXIDATION OF 5-ENOLPYRUVOYLSHIKIMATE-3-PHOSPHATE SYNTHASE FROM ESCHERICHIA-COLI - EVIDENCE FOR A REACTIVE IMIDAZOLE GROUP (HIS385) AT THE HERBICIDE GLYPHOSATE-BINDING SITE

被引:5
作者
HUYNH, QK
机构
[1] Department of Protein Biochemistry, Monsanto Corporate Research, The Monsanto Company, St Louis
关键词
D O I
10.1042/bj2900525
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Photo-oxidation of Escherichia coli 5-enolpyruvoylshikimate-3-phosphate synthase, a target for the non-selective herbicide glyphosate (N-phosphonomethylglycine), in the presence of pyridoxal 5'-phosphate resulted in irreversible inactivation of the enzyme. The inactivation followed pseudo-first-order and saturation kinetics with a K(inact.) of 50 muM. The inactivation is specifically prevented by preincubation of the enzyme with the combination of shikimate 3-phosphate and glyphosate. Increasing glyphosate concentration during preincubation resulted in a decreasing rate of inactivation. On 95 % inactivation, approximately one histidine per molecule of enzyme was oxidized. Tryptic mapping of the enzyme modified in the absence and presence of shikimate 3-phosphate and glyphosate as well as analyses of the histidine content in the isolated peptides indicated that His385, in the peptide Asn383-Asp-His-Arg386, was the site of oxidation. These results suggest that His385 is the most accessible reactive imidazole group under these conditions and is located close to the glyphosate-binding site.
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页码:525 / 530
页数:6
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