Pulsed EPR analysis of tartrate dehydrogenase active-site complexes.
Mn2+.tartrate dehydrogenase.substrate complexes have been examined by electron spin echo envelope modulation spectroscopy. The occurrence of dipolar interactions between Mn2+ and 2H on [2H]pyruvate and [4-2H]NAD(H) confirms that Mn2+ binds at the enzyme active site. The 2H signal arising from labeled pyruvate was lost if the sample was incubated at room temperature, indicating that the enzyme catalyzes exchange between the pyruvate methyl protons and solvent protons. Mn-133Cs dipolar coupling was also observed, which suggests that the monovalent cation cofactor also binds in the active site. The tartrate analogue oxalate was observed to have a significant effect on the binding of NAD(H). Oxalate appears to constrain the binding of NAD(H) so that the nicotinamide portion of the cofactor is held in close proximity to Mn2+. Spectra of enzyme complexes prepared with (R)-[4-2H]NADH showed a more intense 2H signal than analogous complexes prepared with (S)-[4-2H]NADH, demonstrating that the pro-R position of NADH is closer to Mn2+ than the pro-S position and suggesting that tartrate dehydrogenase is an A-side-specific dehydrogenase. Oxalate also affected Cs+ binding; the intensity of the 133Cs signal increased in the presence of oxalate, which suggest that oxalate facilitates binding of Cs+ to the active site or that Cs+ binds closer to Mn2+ when oxalate is present. In addition to signals from substrates, electron spin echo envelope modulation spectra revealed 14N signals that arose from coordination to Mn2+ by nitrogen-containing ligands from the protein; however, the identity of this ligand or ligands remains obscure.
Tipton
P A
PA
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461.
Peisach
J
J
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D001665:Binding Sites; D002586:Cesium / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006859:Hydrogen / Q000378:metabolism; D008024:Ligands; D008345:Manganese / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009584:Nitrogen / Q000737:chemistry; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity
D008024:Ligands; D009243:NAD; D002586:Cesium; D008345:Manganese; D006859:Hydrogen; D000429:Alcohol Oxidoreductases; C038481:tartrate dehydrogenase; D009584:Nitrogen