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Impact of the E57D and N189L mutations on catalysis of M. tuberculosis IGPS
Author(s) -
Cho Sarah
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.05424
Subject(s) - enzyme kinetics , chemistry , mutant , stereochemistry , kinetics , indole test , mycobacterium tuberculosis , catalysis , ligand (biochemistry) , wild type , enzyme , active site , biochemistry , tuberculosis , receptor , medicine , physics , pathology , quantum mechanics , gene
Indole‐3‐glycerol phosphate synthesis (IGPS) is an enzyme that catalyzes the ring closure in 1‐( o ‐carbocyphenylamino)‐1‐deoxyribulose 5‐phosphate (CdRP). Multiple steps are expected to be involved in formation of the pyrrole ring including dehydration, decarboxylation, cyclization, and condensation. IGPS is an essential protein in the pathogen Mycobacterium tuberculosis and a potential target in the treatment of drug‐resistant tuberculosis. In order to better understand the function of Mycobacterium tuberculosis IGPS (MtIGPS), we introduced single‐point mutations into active site residues. I expressed and purified MtIGPS mutants N189L and E57D. The catalytic activities of the mutants were lower than that of the wild type MtIGPS, indicating that these residues play important roles in catalysis. The Michaelis constant, pH‐dependence, and temperature dependence of the mutants were also determined. Wildtype MtIGPS was found to have a K m of 3.0 ± 0.6uM and a k cat value of 1.8 ± 0.13s ‐1 whereas E57D kinetics revealed a K m of 11.6 ± 2.1 uM and a k cat value of 0.047 ± 0.0009s ‐1 . N189L indicated no measurable activity and could not be further characterized. The data on E57D show an altered pH profile, possibly resulting from the lower p Ka of D compared to E, and support the hypothesis that E57 serves as the catalytic base in MtIGPS mechanism. Together these data contribute to an improved understanding of the MtIGPS catalysis and ligand binding.