Structural and functional linkages between subunit interfaces in mammalianpyruvate kinase

Mammalian pyruvate kinase (PK) is a four-domain enzyme that is active as a homo-tetramer. Tissue-specific isozymes of PK exhibit distinct levels of al losteric regulation. PK expressed in muscle tissue (M-1-PK) shows hyperboli c steady-state kinetics, whereas PK expressed in kidney tissue (M-2-PK) dis plays sigmoidal kinetics. Rabbit M-1 and M-2-PK are isozymes whose sequence s differ in only 22 out of 530 residues per subunit, and these changes are localized in an inter-subunit interface. Previous studies have shown that a single amino acid mutation to M-1-PK at either the Y (S402P) or Z (T340 M) subunit interface can confer a level of allosteric regulation that is inte rmediate to M-1-PK and M-2-PK. In an effort to elucidate the roles of the i nter-subunit interaction in signal transmission and the functional/structur al connectivity between these interfaces, the S402P mutant of M-1-PK was cr ystallized and its structure resolved to 2.8 Angstrom. Although the overall S402P M-1-PK structure is nearly identical with the wild-type structure wi thin experimental error, significant differences in the conformation of the backbone are found at the site of mutation along the Y interface. In addit ion, there is a significant change along the Z interface, namely, a loss of an inter-subunit salt-bridge between Asp177 of domain B and Arg341 of doma in A of the opposing subunit. Concurrent with the loss of the salt-bridge i s an increase in the degree of rotational flexibility of domain B that cons titutes the active site. Comparison of previous PK structures shows a corre lation between an increase in this domain movement with the loss of the Asp 177: Arg341 salt-bridge. These results identify the structural linkages bet ween the Y and Z interfaces in regulating the interconversion of conformati onal states of rabbit M-1-PK. (C) 2001 Academic Press.