Effects of primary sequence differences on the global structure and function of an enzyme: a study of pyruvate kinase isozymes.
Pyruvate kinase is an important glycolytic enzyme which is expressed differentially as four distinct isozymes whose catalytic activity is regulated in a tissue-specific manner. The kidney isozyme is known to exhibit sigmoidal kinetics, whereas the muscle isozyme exhibits hyperbolic kinetic properties. By integration of the crystallographic [Stuart, D. I., Levine, M., Muirhead, H., & Stammers, D.K. (1979) J. Mol. Biol. 134, 109-142] and primary sequence data [Noguchi, T., Inoue, H., & Tanaka, T. (1986) J. Biol. Chem. 261, 13807], it was shown that the primary sequence for the C alpha 1 and C alpha 2 regions may constitute the allosteric switching site. To provide insights into the effects of the localized sequence change on the global structural and functional behavior of the enzyme, kinetic studies under a wide spectrum of conditions were conducted for both the muscle and kidney isozymes. These conditions include measurements of enzyme activity as a function of substrate concentrations with different concentrations of allosteric inhibitors or activators. These results showed that both isozymes exhibit the same regulatory properties although quantitatively the distribution of active and inactive forms and the various dissociation constants which govern the binding of substrate and allosteric effectors with the enzyme are different. For such a majority of equilibrium constants to be altered, the localized primary sequence change must confer global perturbations which are manifested as differences in the various equilibrium constants. Structural information about these two isozymes was provided by phase-modulation measurement of the fluorescence lifetime of tryptophan residues under a variety of experimental conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
Consler
T G
TG
E. A. Doisy Department of Biochemistry, St. Louis University School of Medicine, Missouri 63104.
Woodard
S H
SH
Lee
J C
JC
D000818:Animals; D005453:Fluorescence; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D011770:Pyruvate Kinase / Q000378:metabolism*; D011817:Rabbits; D013329:Structure-Activity Relationship
D007527:Isoenzymes; D011770:Pyruvate Kinase