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 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 Five single mutants have now been made in which each of the Trp residues in turn has been replaced by Phe.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 20.8 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, were recorded at both pH 7 and 8.8, and four of the mutants showed essentially the same perturbation in the 280 nm region as the wild-type enzyme. W64F, however, showed essentially no change. W64 is thus clearly a passive reporter of the pH-dependent conformational change, and not actually required for the transition to occur. The CD comparisons also suggest that the aromatic CD spectrum is contributed almost entirely by W64 and W449. Consistent with the pH-dependent change, all five mutant proteins also showed a positively cooperative response to glutamate at pH 9, reversing the inactivation. However, the Hill coefficient decreased from > 5 for wild-type to approximately 3 for the active site cleft mutation W243F and to approximately 2 for the interfacial mutants W64F and W449F in which the trimer-trimer interaction may be directly interrupted. W64 of each subunit is in contact with W449 in its dimer partner at the trimer-trimer interface. It seems that, although neither of these two residues is required for the pH-dependent change, together, they are essential in mediating the total cooperativity of the hexameric enzyme's response to glutamate and are presumably directly involved in transmitting conformational information between the two trimers.
Hamza
Muaawia A
MA
School of Biomolecular and Biomedical Science, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Ireland.
Martin
Stephen R
SR
Engel
Paul C
PC
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D048009:Clostridium symbiosum / Q000201:enzymology*; D004795:Enzyme Stability; D005969:Glutamate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D018698:Glutamic Acid; D010649:Phenylalanine; D014364:Tryptophan; D005969:Glutamate Dehydrogenase