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Modification of a catalytically important residue of indoleglycerol‐phosphate synthase from Escherichia coli
Author(s) -
Eberhard Marc,
Kirschner Kasper
Publication year - 1989
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(89)80225-x
Subject(s) - escherichia coli , active site , residue (chemistry) , site directed mutagenesis , chemistry , serine , enzyme , biochemistry , atp synthase , phosphate , stereochemistry , mutant , binding site , mutagenesis , gene
The active‐site residues of indoleglycerol‐phosphate synthase from Escherichia coli were tentatively localized by comparing crystallographic data with the amino acid identities among the known indoleglycerol‐phosphate synthase sequences. To test the validity of the resulting model of catalysis one of the residues in the presumptive active site, Lys 55, was changed to serine using oligonucleotide‐directed mutagenesis. The specificity constant k cat / K m of the mutant is 3 × 10 4 ‐times lower than that of the wild‐type enzyme, due to a 60‐fold decrease in k cat and a 450‐fold increase in K m . This finding shows that Lys 55 is important for both catalysis and substrate binding.