Allostery in rabbit pyruvate kinase: development of a strategy to elucidate the mechanism

Isozymes of pyruvate kinase (PK) expressed in rabbit muscle and kidney show different allosteric kinetics. The only amino acid changes in the two isozymes, originating from alternative RNA splicing, occur at a stretch of 55 amino acids in the C domain near the subunit interface. The self-correcting distance geometry (SECODG) program DIAMOD was used to calculate a homology model of these interfacial contacts in the four helix bundle of the kidney PK dimer, based on the X-ray structure of the tetrameric rabbit muscle PK [Larsen et al. (1994) Biochemistry 33, 6301-6309]. Energy refinement with the program FANTOM, using the ECEPP/2 force field to assess packing and electrostatic interactions between the two subunits, yielded two groups of energetically favorable conformations. The primary difference in the two groups is the loop conformation of residue Pro 402, which is serine in muscle PK. In one loop conformation, the conserved Lys 421 can form an intersubunit salt bridge as observed in the muscle PK crystal structure. The other loop conformation favors an alternative intrasubunit salt bridge, similar to that found in the Escherichia coli PK structure, which was not used for generating the model. The intersubunit salt bridge leads to an intersubunit hydrogen bonding between Lys 421 of one subunit and Tyr 443 of the other. To provide direct evidence on the roles of these residues, site-directed mutagenesis of the muscle PK gene was conducted. Converting Ser 402 to a proline and Tyr 443 to a phenylalanine changed neither the secondary nor the tetrameric structure, as measured by far UV-CD and sedimentation velocity, respectively. However, the S402P mutant exhibits steady-state kinetics, indicating that the mutant is more reponsive to regulation by effectors, while the mutant Y443F was essentially equivalent to wild-type muscle PK protein except for a lower affinity to phosphoenolpyruvate. These findings suggest a pivotal role for a few key residues in the allosteric regulation in PK.