Significance of mutations on the structural perturbation of thymidylate synthase: Implications for their involvement in subunit exchange

• Published in: Protein science Vol. 16; no. 7; pp. 1439 - 1448
• Main Authors: Saxl, Ruth L., Maley, Gladys F., Hauer, Charles R., Maccoll, Robert, Changchien, Liming, Maley, Frank
• Format: Journal Article
• Language: English
• Published: Bristol Cold Spring Harbor Laboratory Press 01.07.2007
• Subjects: Dimerization; Enzyme Stability - drug effects; Escherichia coli; Hydrogen-Ion Concentration; Ligands; Mass Spectrometry; Models, Molecular; Mutation; mutations; peptide identification; Protein Structure, Quaternary; Protein Structure, Tertiary; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - metabolism; proteolysis; Structure-Activity Relationship; subunit dissociation; thymidylate synthase; Thymidylate Synthase - chemistry; Thymidylate Synthase - genetics; Thymidylate Synthase - metabolism; Trypsin - metabolism; Ultracentrifugation; Urea - pharmacology
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.062509807

Wild‐type thymidylate synthase (WT‐TS) from Escherichia coli and several of its mutants showed varying degrees of susceptibility to trypsin. While WT‐TS was resistant to trypsin as were the mutants C146S, K48E, and R126K, others such as Y94A, Y94F, C146W, and R126E were digested but at different rates from one another. The peptides released from the mutants were identified by mass spectrometry and Edman sequence analysis. The known crystal structures for WT‐TS, Y94F, and R126E, surprisingly, showed no structural differences that could explain the difference in their susceptibility to trypsin. One explanation is that the mutations could perturb the dynamic equilibrium of the dimeric state of the mutants as to increase their dissociation to monomers, which being less structured than the dimer, would be hydrolyzed more readily by trypsin. Earlier studies appear to support this proposal since conditions that promote subunit dissociation in solutions of R126E with other inactive mutants, such as dilution, low concentrations of urea, and elevated pH, greatly enhance the rate of restoration of TS activity. Analytic ultracentrifuge studies with various TSs in urea, or at pH 9.0, or that have been highly diluted are, for the most part, in agreement with this thesis, since these conditions are associated with an increase in dissociation to monomers, particularly with the mutant TSs. However, these studies do not rule out the possibility that conformation differences among the various TS dimers are responsible for the differences in susceptibility to trypsin, particularly at high concentrations of protein where the WT‐TS and mutants are mainly dimers.