Journal
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Volume 397, Issue 1, Pages 28-39Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1006/abbi.2001.2634
Keywords
yeast pyruvate kinase; site-specific mutagenesis; allosteric regulation; subunit interface
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The mechanism by which pyruvate kinase (PK) is allosterically activated by fructose-1,6-bisphosphate (FBP) is poorly understood. To identify residues key to allostery of yeast PK, a point mutation strategy was used. T403E and R459Q mutations in the FBP binding site caused reduced FBP affinity. Introducing positive charges at the 403, 458, and 406 positions in the FBP binding site had little consequence. The mutation Q299N in the A-A subunit interface caused the enzyme response to ADP to be sensitive to FBP. The T311M A-A interface mutant has a decreased affinity for PEP and FBP, and is dependent on FBP for activity. The R369A mutation in the C-C interface only moderately influenced allostery. Creating an E392A mutation in the C-C subunit interface eliminated all cooperativity and allosteric regulation. None of the seven A-C domain interface mutations altered allostery. A model that includes a central role for E392 in allosteric regulation of yeast PK is proposed. (C) 2001 Elsevier Science.
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