Structure of the oxalate-ATP complex with pyruvate kinase: ATP as a bridging ligand for the two divalent cations
The 2 equiv of divalent cation that are required cofactors for pyruvate kinase reside in sites of different affinities for different species of cation. The intrinsic selectivity of the protein-based site for Mn(II) and of the nucleotide-based site for Mg(II) has been exploited in electron paramagnetic resonance (EOR) investigations of ligands for Mn(II) at the protein-based site. Oxalate, a structural analogue of the enolate of pyruvate, has been used as a surrogate for the reactive form of pyruvate in complexes with enzyme, Mn(II), Mg(II), and ATP. Superhyperfine coupling between the unpaired electron spin of Mn(II) and the nuclear spin of /sup 17/O, specifically incorporated into oxalate, shows that oxalate is bound at the active site as a bidentate chelate with Mn(II). Coordination of the ..gamma..-phosphate of ATP to this same Mn(II) center is revealed by observation of superhyperfine coupling from /sup 17/O regiospecifically incorporated into the ..gamma..-phosphate group of ATP. By contrast, /sup 17/O in the ..cap alpha..-phosphate or in the ..beta..-phosphate groups of ATP does not influence the spectrum. Experiments in /sup 17/O-enriched water show that there is also a single water ligand bound to the Mn(II). These data indicate that ATP bridges Mn(II) and Mg(II) at the active site. A close spacing of the two divalent cations is also evident from the occurrence of magnetic interactions for complexes in which 2 equiv of Mn(II) are present at the active site. The structure for the enzyme-Mn(II)-oxalate-Mg(II)-ATP complex suggests a scheme for the normal reverse reaction of pyruvate kinase in which the divalent cation at the protein-based site activates the keto acid substrate through chelation and promotes phospho transfer by simultaneous coordination to the enolate oxygen and to a pendant oxygen from the ..gamma..-phosphate of ATP.
- Research Organization:
- Univ. of Pennsylvania School of Medicine, Philadelphia
- OSTI ID:
- 5514810
- Journal Information:
- Biochemistry; (United States), Vol. 26:8
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
ATP
ELECTRON SPECTRA
PHOSPHOTRANSFERASES
CHEMICAL ACTIVATION
CATIONS
COMPLEXES
ELECTRON SPIN RESONANCE
HYPERFINE STRUCTURE
LABELLED COMPOUNDS
LIGANDS
MAGNESIUM COMPOUNDS
MANGANESE COMPOUNDS
OXALATES
OXYGEN 17
PYRUVIC ACID
WATER
ALKALINE EARTH METAL COMPOUNDS
CARBOXYLIC ACID SALTS
CARBOXYLIC ACIDS
CHARGED PARTICLES
ENZYMES
EVEN-ODD NUCLEI
HYDROGEN COMPOUNDS
IONS
ISOTOPES
KETO ACIDS
LIGHT NUCLEI
MAGNETIC RESONANCE
NUCLEI
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
OXYGEN COMPOUNDS
OXYGEN ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
RESONANCE
SPECTRA
STABLE ISOTOPES
TRANSFERASES
TRANSITION ELEMENT COMPOUNDS
550201* - Biochemistry- Tracer Techniques