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.