Characterization of tryptophan-shifted mutants of Phosphofructokinase from Bacillus stearothermophilus /
Phosphofructokinase isolated from Bacillus stearothermophilus (BsPFK) is a homotetrameric enzyme which is allosterically regulated by PEP (inhibitor) and MgADP (activator). Each subunit of BsPFK has a single tryptophan residue which in principle could be used to examine the biophysical properties o...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
2000.
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| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=728408671&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Phosphofructokinase isolated from Bacillus stearothermophilus (BsPFK) is a homotetrameric enzyme which is allosterically regulated by PEP (inhibitor) and MgADP (activator). Each subunit of BsPFK has a single tryptophan residue which in principle could be used to examine the biophysical properties of the enzyme using fluorescence spectroscopy. However, the fluorescence of the tryptophan in its native position is not responsive to ligand binding. In an attempt to circumvent the inadequacy of tryptophan as a fluorescence probe in its native position, several tryptophan-shifted mutants have been constructed. These mutants have kinetic and allosteric properties that are similar to the wild-type enzyme as assessed using steady-state activity assays; however, each of the mutants displays unique fluorescence characteristics. The fluorescence properties of these mutants have been exploited to examine the biophysical characteristics of BsPFK under equilibrium conditions. Specifically, W179F/F230W and W179F/F139W have been useful in examining the allosteric interaction between Fru-6-P and PEP under equilibrium conditions. In both cases, the extent of antagonism between these ligands is the same regardless of whether steady-state kinetic or steady-state fluorescence techniques are used suggesting that the rapid equilibrium assumption as it pertains to Fru-6-P and PEP binding is valid and that MgATP does not influence the coupling between Fru-6-P and PEP. Furthermore, homotropic cooperativity for the allosteric effector PEP has been demonstrated. These mutants were also used to examine the dynamics of the local tryptophan environment. For the areas probed, BsPFK appears to have a rigid structure and the flexibility of the local environment was not affected by ligand binding to a significant extent. Overall, these mutants have provided a useful means to examine the biophysical properties of BsPFK under equilibrium conditions using fluorescence spectroscopy. Most of the substitutions examined were rather conservative in that they did not alter the function of the enzyme, so the results obtained can be directly related to the wild-type enzyme. |
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| Item Description: | Vita. "Major Subject: Biochemistry". |
| Physical Description: | xxi, 208 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 196-207). |