Persistent Binding of MgADP to the E187A Mutant of Escherichia coli Phosphofructokinase in the Absence of Allosteric Effects
MgADP binding to the allosteric site enhances the affinity of Escherichia coli phosphofructokinase (PFK) for fructose 6-phosphate (Fru-6-P). X-ray crystallographic data indicate that MgADP interacts with the conserved glutamate at position 187 within the allosteric site through an octahedrally coord...
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Published in | Biochemistry (Easton) Vol. 40; no. 13; pp. 4140 - 4149 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
Published |
United States
American Chemical Society
03.04.2001
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Subjects | |
Online Access | Get full text |
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Summary: | MgADP binding to the allosteric site enhances the affinity of Escherichia coli phosphofructokinase (PFK) for fructose 6-phosphate (Fru-6-P). X-ray crystallographic data indicate that MgADP interacts with the conserved glutamate at position 187 within the allosteric site through an octahedrally coordinated Mg2+ ion [Shirakihara, Y., and Evans, P. R. (1988) J. Mol. Biol. 204, 973−994]. Lau and Fersht reported that substituting an alanine for this glutamate within the allosteric site of PFK (i.e., mutant E187A) causes MgADP to lose its allosteric effect upon Fru-6-P binding [Lau, F. T.-K., and Fersht, A. R. (1987) Nature 326, 811−812]. However, these authors later reported that MgADP inhibits Fru-6-P binding in the E187A mutant. The inhibition presumably occurs by preferential binding to the inactive (T) state complex of the Monod−Wyman−Changeux two-state model [Lau, F. T.-K., and Fersht, A. R. (1989) Biochemistry 28, 6841−6847]. The present study provides an alternative explanation of the role of MgADP in the E187A mutant. Using enzyme kinetics, steady-state fluorescence emission, and anisotropy, we performed a systematic linkage analysis of the three-ligand interaction between MgADP, Fru-6-P, and MgATP. We found that MgADP at low concentrations did not enhance or inhibit substrate binding. Anisotropy shows that MgADP binding at the allosteric site occurred even when MgADP produced no allosteric effect. However, as in the wild-type enzyme, the binding of MgADP to the active site in the mutant competitively inhibited MgATP binding and noncompetitively inhibited Fru-6-P binding. These results clarified the mechanism of a three-ligand interaction and offered a nontraditional perspective on allosteric mechanism. |
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Bibliography: | This work was performed at Texas A&M University in partial fulfillment of the dissertation for the Ph.D. in Biochemistry and was supported by Grant GM-33216 from the National Institutes of Health. ark:/67375/TPS-K48PB4QD-D istex:CCE5F9B90C7789DB70ECB3490F41398A02D2EEC6 ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 |
ISSN: | 0006-2960 1520-4995 |
DOI: | 10.1021/bi001768z |