Interfacial communications in recombinant rabbit kidney pyruvate kinase.
Tissue-specific isozymes of pyruvate kinase are particularly attractive systems to elucidate the molecular mechanism(s) of conferring allostery. The muscle- and kidney-type isozymes are coded by the same gene. As a consequence of alternative message RNA splicing, the two primary sequences differ by a small number of residues. However, they exhibit very 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 coli. The primary structure of recombinant rabbit kidney-type pyruvate kinase (rRKPK) and recombinant rabbit muscle-type pyruvate kinase (rRMPK) differ at 22 positions, which are located in a region that forms important intersubunit contacts in the RMPK structure. Velocity sedimentation and analytical gel chromatographic studies show that rRKPK undergoes reversible dimer left and right arrow tetramer assembly with an equilibrium constant of 28 +/- 3 mL/mg. This subunit assembly process provides the opportunity to elucidate the role of this dimer interface in transmission of signal upon binding of substrates and allosteric effectors. The assembly to tetrameric rRKPK is favored by the binding of phosphoenolpyruvate (PEP), one of the two substrates, or fructose 1,6-bisphosphate (FBP), an activator. In contrast, the equilibrium is shifted toward dimeric rRKPK upon binding of adenosine diphosphate (ADP), the other substrate, or l-phenylalanine (Phe), the inhibitor. These observations provide significant new insights to the molecular mechanism of allosteric regulation in the pyruvate kinase system. First, all substrates and effectors communicate through this particular dimer-dimer interface. Second, the thermodynamic signatures of these communications are qualitatively different for the two substrates and between the activator, FBP, and inhibitor, Phe.
Friesen
R H
RH
Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1055, USA.
Chin
A J
AJ
Ledman
D W
DW
Lee
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011770:Pyruvate Kinase / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D007527:Isoenzymes; D011994:Recombinant Proteins; D011770:Pyruvate Kinase