A Solution NMR and Crystallographic Study of the Role of the Quaternary Shift in the Allosteric Regulation of Phosphofructokinase from B. stearothermophilus

Abstract

The 136 kDa Bacillus stearothermophilus phosphofructokinase (BsPFK) is a homotetramer that is allosterically inhibited by phospho(enol)pyruvate (PEP), which binds along one dimer-dimer interface. Fru-6-P binds along the other dimer-dimer interface. The substrate bound and the inhibitor bound structures of wild-type BsPFK exhibit a 7° rotation about the substrate binding site interface, termed the quaternary shift. The binding of substrate and inhibitor to BsPFK have been studied using methyl TROSY NMR. By selectively labeling all 30 Ile residues in the BsPFK monomer with 13CH3 in an otherwise fully deuterated enzyme, only the Ile are detected on a 2D-1H-13C correlation spectrum. Several distinct Ile cross-peaks change position when PEP is added to wild-type apo BsPFK. To distinguish between changes associated with the quaternary shift and those associated with intra-subunit tertiary changes, the variant D12A BsPFK is currently being studied using kinetics, x-ray crystallography, and methyl TROSY NMR. When compared to wild-type, D12A BsPFK shows a 100-fold increase in the binding affinity for PEP, a 50-fold decrease in the binding affinity for F6P, and an allosteric coupling comparable to wild-type. Crystal structures of apo and PEP bound forms of D12A BsPFK both indicate a shifted structure similar to the inhibitor bound structure of wild-type. Since PEP still inhibits D12A BsPFK substantially despite the fact that it has already adopted the inhibited quaternary structure, the inhibition likely involves further tertiary changes to the enzyme structure. NMR of deuterated, 13CH3 labeled D12A is being performed in an effort to identify residues involved in these tertiary changes. Supported by grant GM33216 from NIH and grant A1543 from the Welch Foundation.