INTERFACIAL COMMUNICATIONS IN RECOMBINANT RABBIT KIDNEY PYRUVATE-KINASE

Tissue-specific isozymes of pyruvate kinase are particularly attractiv e systems to elucidate the molecular mechanism(s) of conferring allost ery. The muscle-and kidney-type isozymes are coded by the same gene. A s a consequence of alternative message RNA splicing, the two primary s equences differ by a small number of residues. However, they exhibit v ery different regulatory behavior. In an effort to identify the roles of specific residues in conferring allostery, the gene encoding rabbit kidney-type pyruvate kinase was cloned and expressed in Escherichia c oli. The primary structure of recombinant rabbit kidney-type-pyruvate kinase (rRKPK) and recombinant rabbit muscle-type pyruvate kinase (rRM PK) differ at 22 positions, which are located in a region that forms i mportant intersubunit contacts in the RMPK structure. Velocity sedimen tation and analytical,eel chromatographic studies show that rRKPK unde rgoes reversible dimer <-> tetramer assembly with an equilibrium const ant of 28 +/- 3 mL/mg. This subunit assembly process provides the oppo rtunity to elucidate the role of this dimer interface in transmission of signal upon binding of substrates and allosteric effecters. The ass embly to tetrameric rRKPK is favored by the binding of phosphoenolpyru vate (PEP), one of the two substrates, or fructose 1,6-bisphosphate (F BP), an activator. In contrast, the equilibrium is shifted toward dime ric rRKPK upon binding of adenosine diphosphate (ADP), the ether subst rate, or L-phenylalanine (Phe), the inhibitor. These observations prov ide significant new insights to the molecular mechanism of allosteric regulation in the pyruvate kinase system. First, all substrates and ef fecters communicate through this particular dimer-dimer interface. Sec ond, the thermodynamic signatures of these communications are qualitat ively different for the two substrates and between the activator, FBP, and inhibitor, Phe.