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Substrate induced structural and dynamics changes in human phosphomevalonate kinase and implications for mechanism

Phosphomevalonate kinase (PMK) catalyzes an essential step in the mevalonate pathway, which is the only pathway for synthesis of isoprenoids and steroids in humans. PMK catalyzes transfer of the γ‐phosphate of ATP to mevalonate 5‐phosphate (M5P) to form mevalonate 5‐diphosphate. Bringing these phosphate groups in proximity to react is especially challenging, given the high negative charge density on the four phosphate groups in the active site. As such, conformational and dynamics changes needed to form the Michaelis complex are of mechanistic interest. Herein, we report the characterization of substrate induced changes (Mg‐ADP, M5P, and the ternary complex) in PMK using NMR‐based dynamics and chemical shift perturbation measurements. Mg‐ADP and M5P Kd's were 6–60 μM in all complexes, consistent with there being little binding synergy. Binding of M5P causes the PMK structure to compress (τc = 13.5 nsec), whereas subsequent binding of Mg‐ADP opens the structure up (τc = 15.6 nsec). The overall complex seems to stay very rigid on the psec‐nsec timescale with an average NMR order parameter of S2 ∼0.88. Data are consistent with addition of M5P causing movement around a hinge region to permit domain closure, which would bring the M5P domain close to ATP to permit catalysis. Dynamics data identify potential hinge residues as H55 and R93, based on their low order parameters and their location in extended regions that connect the M5P and ATP domains in the PMK homology model. Likewise, D163 may be a hinge residue for the lid region that is homologous to the adenylate kinase lid, covering the “Walker‐A” catalytic loop. Binding of ATP or ADP appears to cause similar conformational changes; however, these observations do not indicate an obvious role for γ‐phosphate binding interactions. Indeed, the role of γ‐phosphate interactions may be more subtle than suggested by ATP/ADP comparisons, because the conservative O to NH substitution in the β‐γ bridge of ATP causes a dramatic decrease in affinity and induces few chemical shift perturbations. In terms of positioning of catalytic residues, binding of M5P induces a rigidification of Gly21 (adjacent to the catalytically important Lys22), although exchange broadening in the ternary complex suggests some motion on a slower timescale does still occur. Finally, the first nine residues of the N‐terminus are highly disordered, suggesting that they may be part of a cleavable signal or regulatory peptide sequence. Proteins 2009. © 2008 Wiley‐Liss, Inc.