Thermodynamic, fluorescence dynamic, and kinetic characterization of the E187A mutant of Escherichia coli phosphofructokinase /
A thermodynamic linked-function approach is utilized to assess the possible coupling interactions between substrates and allosteric legends toward the E187A mutant of E. coli phosphofructokinase. The mutation of G1u-187 to an alumine was reported to induce activation by phosphoenolpyruvate, a wild-t...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
1999.
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| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=730294041&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | A thermodynamic linked-function approach is utilized to assess the possible coupling interactions between substrates and allosteric legends toward the E187A mutant of E. coli phosphofructokinase. The mutation of G1u-187 to an alumine was reported to induce activation by phosphoenolpyruvate, a wild-type inhibitor, and at the same time abolish the allosteric activity by MgADP, a wild- type activator. This mutant has provided us with useful insights into the mechanisms of allosteric phenomena, specifically the relationship between binding events, linked interactions, and structure-dynamic relationships. Residue 187 of the allosteric site does not participate in binding but rather assumes a predominant role in allosteric regulation. The binding of a particular ligand without influence by other legends is relatively indistinguishable from the wild-type enzyme. However, the action by a particular ligand is drastically altered by the presence second or third ligand. MgADP becomes unresponsive toward Fru-6P, PEP becomes a weak activator toward Fru-6P, and MgATP mitigates PEP'S inhibition toward Fru-6P and even induces activation in higher concentrations. In addition, we were able to discriminate the differential responsiveness of the intrinsic tryptophanyl probe solely attributed to modified allosteric ligand binding events rather than to mutation-induced fluorescence alterations. Dynamic fluorescence characterization of the tryptophanyl lifetime provides evidence for increased heterogeneity and flexible local environment. Linked interactions in the pre-steady-state realm indicate that allosteric effectors influence certain intrinsic rate constants for binding, predominantly the rate constant describing the complication of the ligand to the enzyme to form an enzyme-ligand intermediate complex. The thermodynamic strategy elucidates several key aspects of linkage. First, although MgADP ceases to enhance Fru-6P, binding of MgADP and linkage interaction to Fru-6P is evident from the dynamic lifetime and transient kinetic studies. Second, the activation by PEP toward Fru-6P is related to the formation the quaternary interaction completed by MgATP or AMPPCP. Third, similar to the enthalpy-entropy linked interaction, the intrinsic kinetic Modifications resulting from a coupling interaction or due to the mutation appeared compensatory. These results taken altogether reflect the hierarchical continuity of protein structure and energetic, which manifest in the interactions between each ligand to the protein and multiple legends toward each other resulting in the phenomenology of allosteric effects. |
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| Item Description: | Vita. In title numerals are used. "Major Subject: Biochemistry". |
| Physical Description: | xxii, 271 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 256-270). |