Mycobacterium tuberculosis ketopantoate hydroxymethyltransferase: tetrahydrofolate-independent hydroxymethyltransferase and enolization reactions with alpha-keto acids.
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.
Sugantino
Michele
M
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Zheng
Renjian
R
Yu
Michael
M
Blanchard
John S
JS
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*
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