Structural and functional linkages between subunit interfaces in mammalian pyruvate 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 allosteric regulation. PK expressed in muscle tissue (M1-PK) shows hyperbolic steady-state kinetics, whereas PK expressed in kidney tissue (M2-PK) displays sigmoidal kinetics. Rabbit M1 and M2-PK are isozymes whose sequences 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 M1-PK at either the Y (S402P) or Z (T340 M) subunit interface can confer a level of allosteric regulation that is intermediate to M1-PK and M2-PK. In an effort to elucidate the roles of the inter-subunit interaction in signal transmission and the functional/structural connectivity between these interfaces, the S402P mutant of M1-PK was crystallized and its structure resolved to 2.8 A. Although the overall S402P M1-PK structure is nearly identical with the wild-type structure within experimental error, significant differences in the conformation of the backbone are found at the site of mutation along the Y interface. In addition, 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 domain A of the opposing subunit. Concurrent with the loss of the salt-bridge is an increase in the degree of rotational flexibility of domain B that constitutes the active site. Comparison of previous PK structures shows a correlation between an increase in this domain movement with the loss of the Asp177: Arg341 salt-bridge. These results identify the structural linkages between the Y and Z interfaces in regulating the interconversion of conformational states of rabbit M1-PK.
Wooll
J O
JO
Department of Human Biological Chemistry and Genetics and the Sealy Center for Structural Biology, The University of Texas Medical Branch, Galveston, TX 77555-0647, USA.
Friesen
R H
RH
White
M A
MA
Watowich
S J
SJ
Fox
R O
RO
Lee
J C
JC
Czerwinski
E W
EW
D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D019281:Dimerization; D005635:Fructosediphosphates / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000201:enzymology*; D009154:Mutation; D009928:Organ Specificity; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D055672:Static Electricity; D013329:Structure-Activity Relationship
D005635:Fructosediphosphates; D007527:Isoenzymes; D021122:Protein Subunits; D011770:Pyruvate Kinase; C029063:fructose-1,6-diphosphate