title: Use of a purified heterodimer to test negative cooperativity as the basis of substrate inactivation of Escherichia coli thymidylate synthase (Asn177-->Asp).
abstract: BACKGROUND
  Thymidylate synthase (TS) converts deoxyuridylate to thymidylate, an essential DNA precursor. Replacement of Asn177 with aspartate (Asn177-->Asp) in Escherichia coli TS creates a novel ability to methylate 2'-deoxycytidylate (dCMP). The dCMP-methylase activity of TS(Asn177-->Asp) is transiently inactivated by reaction with deoxyuridylate and methylene-tetrahydrofolate, the methyl donor. We have tested the possibility that the inactivation is due to negative cooperativity, created in the TS dimer by the Asn177-->Asp mutation.
  
  
  RESULTS
  A heterodimeric form of TS, containing one wild type and one Asn177-->Asp active site, was created to test for negative cooperativity. Substrate inactivation still occurred, even with the mutation present at only one active site.
  
  
  CONCLUSIONS
  Inactivation of TS(Asn177-->Asp) by deoxyuridylate is not due to negative cooperativity created by the mutation. The 'artificial isozyme' method we have developed for purifying heterodimers away from the progenitor homodimers is generally applicable to other hetero-oligomeric proteins.
authors: Department of Pharmacology, University of Massachusetts Medical Center, Worcester 01605.
authors: L W
authors: LW
authors: Hardy
authors: D F
authors: DF
authors: Pacitti
authors: E
authors: E
authors: Nalivaika
mesh_terms: D000595:Amino Acid Sequence; D001216:Asparagine; D001224:Aspartic Acid; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D046911:Macromolecular Substances; D011994:Recombinant Proteins; D001224:Aspartic Acid; D001216:Asparagine; D013940:Thymidylate Synthase