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.
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