TAILIEUCHUNG - Báo cáo khoa học: The contribution of tryptophan residues to conformational changes in clostridial glutamate dehydrogenase ) W64 and W449 as mediators of the cooperative response to glutamate

The hexameric glutamate dehydrogenase ofClostridium symbiosumhas pre-viously been shown to undergo a pH-dependent inactivating conformational change that perturbs the environment of one or more Trp residues and is reversed by glutamate in a highly cooperative fashion with a Hill coefficient of almost 6. | ễFEBS Journal The contribution of tryptophan residues to conformational changes in clostridial glutamate dehydrogenase - W64 and W449 as mediators of the cooperative response to glutamate Muaawia A. Hamza1 Stephen R. Martin2 and Paul C. Engel1 1 Schoolof Biomolecular and BiomedicalScience Conway Institute of Biomolecular and BiomedicalResearch University College Dublin Ireland 2 Division of PhysicalBiochemistry NationalInstitute for MedicalResearch Mill Hill London UK Keywords allosteric mechanism circular dichroism cooperativity glutamate dehydrogenase tryptophan residues Correspondence P. C. Engel Schoolof Biomolecular and Biomedical Science Conway Institute University College Dublin Belfield Dublin 4 Ireland Fax 353 1283 7211 Tel 353 1716 6764 E-mail Received 13 April2007 revised 30 May 2007 accepted 14 June 2007 doi The hexameric glutamate dehydrogenase of Clostridium symbiosum has previously been shown to undergo a pH-dependent inactivating conformational change that perturbs the environment of one or more Trp residues and is reversed by glutamate in a highly cooperative fashion with a Hill coefficient of almost 6. Five single mutants have now been made in which each of the Trp residues in turn has been replaced by Phe. All five were successfully over-produced as soluble proteins and purified. Far-UV CD showed that none of the mutations significantly affected secondary structure. All five proteins were active ranging from 13 U-mg-1 W64F to U-mg-1 W393F compared to 20 U-mg-1 for wild-type and the kinetic parameters at pH 7 were little changed except for a five- to six-fold increase in Km for glutamate in W243F. Thermostability was also relatively little changed although W310F and W393F were somewhat more stable and W64F less stable than the unmutated enzyme. All still showed the characteristic reversible time-dependent high-pH inactivation. Near-UV CD spectra reflecting the environment of aromatic residues

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