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 th e 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 fo ur helix bundle of the kidney PK dimer, based on the X-ray structure o f the tetrameric rabbit muscle PK [Larsen et al. (1994) Biochemistry 3 3, 6301-6309], Energy refinement with the program FANTOM, using the EC EPP/2 force field to assess packing and electrostatic interactions bet ween the two subunits, yielded two groups of energetically favorable c onformations. The primary difference in the two groups is the loop con formation of residue Pre 402, which is serine in muscle PK. In one loo p conformation, the conserved Lys 421 can form an intersubunit salt br idge as observed in the muscle PK crystal structure. The other loop co nformation favors an alternative intrasubunit salt bridge, similar to that found in the Escherichia coli PK structure, which was not used fo r generating the model. The intersubunit salt bridge leads to an inter subunit 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. Conver ting Ser 402 to a proline and Tyr 443 to a phenylalanine changed neith er the secondary nor the tetrameric structure, as measured by far UV-C D and sedimentation velocity, respectively. However, the S402P mutant exhibits steady-state kinetics, indicating that the mutant is more rep onsive to regulation by effecters, while the mutant Y443F was essentia lly equivalent to wildtype muscle PK protein except for a lower affini ty to phosphoenolpyruvate. These findings suggest a pivotal role for a few key residues in the allosteric regulation in PK.