Multiple turnovers of the nicotino-enzyme PdxB require α-keto acids as cosubstrates.
PdxB catalyzes the second step in the biosynthesis of pyridoxal phosphate by oxidizing 4-phospho-d-erythronate (4PE) to 2-oxo-3-hydroxy-4-phosphobutanoate (OHPB) with concomitant reduction of NAD(+) to NADH. PdxB is a nicotino-enzyme wherein the NAD(H) cofactor remains tightly bound to PdxB. It has been a mystery how PdxB performs multiple turnovers since addition of free NAD(+) does not reoxidize the enzyme-bound NADH following conversion of 4PE to OHPB. We have solved this mystery by demonstrating that a variety of physiologically available α-keto acids serve as oxidants of PdxB to sustain multiple turnovers. In a coupled assay using the next two enzymes of the biosynthetic pathway for pyridoxal phosphate (SerC and PdxA), we have found that α-ketoglutarate, oxaloacetic acid, and pyruvate are equally good substrates for PdxB (k(cat)/K(m) values ~1 × 10(4) M⁻¹s⁻¹). The kinetic parameters for the substrate 4PE include a k(cat) of 1.4 s⁻¹, a K(m) of 2.9 μM, and a k(cat)/K(m) of 6.7 × 10(6) M⁻¹s⁻¹. Additionally, we have characterized the stereochemistry of α-ketoglutarate reduction by showing that d-2-HGA, but not l-2-HGA, is a competitive inhibitor vs 4PE and a noncompetitive inhibitor vs α-ketoglutarate.
Rudolph
Johannes
J
Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, CO 80309, USA
Kim
Juhan
J
Copley
Shelley D
SD
D002237:Carbohydrate Dehydrogenases / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D007651:Keto Acids / Q000378:metabolism*; D007656:Ketoglutaric Acids; D007700:Kinetics; D053858:Metabolic Networks and Pathways; D010071:Oxaloacetates; D016877:Oxidants / Q000378:metabolism; D011732:Pyridoxal Phosphate / Q000096:biosynthesis*; D019289:Pyruvic Acid; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D007651:Keto Acids; D007656:Ketoglutaric Acids; D010071:Oxaloacetates; D016877:Oxidants; D011732:Pyridoxal Phosphate; D019289:Pyruvic Acid; D002237:Carbohydrate Dehydrogenases; C061651:pdxB protein, E coli