Mycobacterium tuberculosis ketopantoate hydroxymethyltransferase: Tetrahydrofolate-independent hydroxymethyltransferase and enolization reactions with α-Keto acids

Michele Sugantino, Renjian Zheng, Michael Yu, John S. Blanchard

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Abstract

The panB gene that encodes ketopantoate hydroxymethyltransferase has been cloned from Mycobacterium tuberculosis, expressed, and purified to homogeneity. 1H NMR spectroscopy was used to determine the rate of (i) tetrahydrofolate-independent hydroxymethyltransferase chemistry between formaldehyde and α-ketoisovalerate and (ii) deuterium exchange in the methylenetetrahydrofolate-independent enolization of α-ketoisovalerate and other α-keto acids, catalyzed by PanB. These studies have demonstrated that substrate enolization by PanB is divalent metal-dependent with a preference of Mg2+ > Zn2+ > Co2+ > Ni2+ > Ca2+. The rate of enolization is pH-dependent with optimal activity in the range of 7.0-7.5. The pH profile was bell-shaped, depending on the ionization state of two ionizable groups with apparent pK values of 6.2 and 8.3. Enolization and isotope exchange occurs with some α-keto acids (e.g., pyruvate and α-ketobutyrate), resulting in the complete exchange of all β-hydrogens. Enzyme-catalyzed enolization and isotope exchange occur with other long-chain and branched α-keto acids, resulting in the stereospecific exchange of only one of the β-hydrogen atoms. These results are discussed in the context of steric restrictions present in the enzyme active site and the stereochemistry of base-catalyzed isotope exchange.

Original languageEnglish (US)
Pages (from-to)191-199
Number of pages9
JournalBiochemistry
Volume42
Issue number1
DOIs
StatePublished - Jan 14 2003

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ASJC Scopus subject areas

  • Biochemistry

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