Structure of the oxalate-ATP complex with pyruvate kinase: ATP as a bridging ligand for the two divalent cations

Journal Article · Tue Apr 21 04:00:00 EDT 1987 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00382a026· OSTI ID:5514810

Lodato, D T; Reed, G H

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), Journal Name: Biochemistry; (United States) Vol. 26:8; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALKALINE EARTH METAL COMPOUNDS
ATP
CARBOXYLIC ACID SALTS
CARBOXYLIC ACIDS
CATIONS
CHARGED PARTICLES
CHEMICAL ACTIVATION
COMPLEXES
ELECTRON SPECTRA
ELECTRON SPIN RESONANCE
ENZYMES
EVEN-ODD NUCLEI
HYDROGEN COMPOUNDS
HYPERFINE STRUCTURE
IONS
ISOTOPES
KETO ACIDS
LABELLED COMPOUNDS
LIGANDS
LIGHT NUCLEI
MAGNESIUM COMPOUNDS
MAGNETIC RESONANCE
MANGANESE COMPOUNDS
NUCLEI
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
OXALATES
OXYGEN 17
OXYGEN COMPOUNDS
OXYGEN ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PYRUVIC ACID
RESONANCE
SPECTRA
STABLE ISOTOPES
TRANSFERASES
TRANSITION ELEMENT COMPOUNDS
WATER