title: Mycobacterium tuberculosis ketopantoate hydroxymethyltransferase: tetrahydrofolate-independent hydroxymethyltransferase and enolization reactions with alpha-keto acids.
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 alpha-ketoisovalerate and (ii) deuterium exchange in the methylenetetrahydrofolate-independent enolization of alpha-ketoisovalerate and other alpha-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 alpha-keto acids (e.g., pyruvate and alpha-ketobutyrate), resulting in the complete exchange of all beta-hydrogens. Enzyme-catalyzed enolization and isotope exchange occur with other long-chain and branched alpha-keto acids, resulting in the stereospecific exchange of only one of the beta-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.
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
authors.forename: Michele
authors.initials: M
authors.lastname: Sugantino
authors.forename: Renjian
authors.initials: R
authors.lastname: Zheng
authors.forename: Michael
authors.initials: M
authors.lastname: Yu
authors.forename: John S
authors.initials: JS
authors.lastname: Blanchard
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002384:Catalysis; D003903:Deuterium / Q000737:chemistry; D004789:Enzyme Activation; D045782:Hemiterpenes; D006863:Hydrogen-Ion Concentration; D019877:Hydroxymethyl and Formyl Transferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D007651:Keto Acids / Q000737:chemistry*; D008670:Metals / Q000737:chemistry; D008968:Molecular Conformation; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010205:Pantothenic Acid / Q000737:chemistry; D011522:Protons; D019289:Pyruvic Acid / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013053:Spectrophotometry; D013379:Substrate Specificity; D013763:Tetrahydrofolates / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D045782:Hemiterpenes; D007651:Keto Acids; D008670:Metals; D011522:Protons; D011994:Recombinant Proteins; D013763:Tetrahydrofolates; C016211:alpha-keto-beta-methylvaleric acid; D010205:Pantothenic Acid; C079776:2-amino-3-ketobutyrate; C001505:alpha-ketoisovalerate; C013082:alpha-ketoisocaproic acid; D019289:Pyruvic Acid; D003903:Deuterium; D019877:Hydroxymethyl and Formyl Transferases; C021477:3-methyl-2-oxobutanoate hydroxymethyltransferase