Photochemically induced dynamic nuclear polarization NMR study of yeast and horse muscle phosphoglycerate kinase

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Abstract
A photochemically induced dynamic nuclear polarization (photo-CIDNP) study of yeast and horse muscle phosphoglycerate kinase with flavin dyes was undertaken to identify the histidine, tryptophan, and tyrosine resonances in the aromatic region of the simplified 1H NMR spectra of these enzymes and to investigate the effect of substrates on the resonances observable by CIDNP. Identification of the CIDNP-enhanced resonances with respect to the type of amino acid residue has been achieved since only tyrosine yields emission peaks and the dye 8-aminoriboflavin enhances tryptophan but not histidine. By use of the known amino acid sequences and structures derived from X-ray crystallographic studies of the enzymes from the two species, assignment of the specific residues in the protein sequences giving rise to the CIDNP spectra was partially achieved. In addition, flavin dye accessibility was used to probe any changes in enzyme structure induced by substrate binding. The nine resonance peaks observed in the CIDNP spectrum of yeast phosphoglycerate kinase have been assigned tentatively to five residues: histidines-53 and -151, tryptophan-310, and tyrosines-48 and -195. The accessibility of a tyrosine to photoexcited flavin is reduced in the presence of MgATP. Since the tyrosine residues are located some distance from the MgATP binding site of the catalytic center, it is proposed that this change is due to a distant conformational change or that a second metal-ATP site inferred from other studies lies close to one of the tyrosines. Horse muscle phosphoglycerate kinase exhibits seven resonances by CIDNP NMR. None of the four tyrosines of the horse enzyme (three are conserved in the yeast enzyme) is observed in the CIDNP spectra. The resonances have been tentatively assigned to tryptophan-310 and to histidines-172, -294, and -390. It is concluded that the conserved residues in the two enzymes do not necessarily have equal accessibility to flavin. Binding of 3-phosphoglycerate to the horse muscle enzyme shifts the resonances assigned to histidine-172 upfield due to either a conformational change of a pKa change of the histidine. The addition of 3-phosphoglycerate and MgATP results in the appearance of two additional resonances in the CIDNP spectrum due to a histidine residue that is inaccessible to flavin in both the enzyme alone and its binary complex with 3-phosphoglycerate. This histidine residue is tentatively assigned to residue 390 which would become accessible to flavin upon loss of the hydrogen bond to glutamic acid residue 192. Weakening of the hydrogen bond between histidine-390 and glutamic acid residue 192 in the hinge region of the bilobal protein structure has been implicated from crystallographic studies as an important factor in the domain movement required to bring the substrates in close enough proximity for direct catalytic transfer of the phosphoryl moiety. The CIDNP spectra are consistent with the suggestions that binding of 3-phosphoglycerate alone is insufficient to effect domain movement and that binding of both substrates are required for conversion of the horse muscle enzyme to its catalytically active form.