USE OF A PURIFIED HETERODIMER TO TEST NEGATIVE COOPERATIVITY AS THE BASIS OF SUBSTRATE INACTIVATION OF ESCHERICHIA-COLI THYMIDYLATE SYNTHASE (ASN177-]ASP)

Background: Thymidylate synthase (TS) converts deoxyuridylate to thymi dylate, an essential DNA precursor. Replacement of Asn177 with asparta te (Asn177->Asp) in Escherichia coil TS creates a novel ability to met hylate 2'-deoxycytidylate (dCMP). The dCMP-methylase activity of TS(As n177->Asp) is transiently inactivated by reaction with deoxyuridylate and methylene-tetrahydrofolate, the methyl doner. We have tested the p ossibility that the inactivation is due to negative cooperativity, cre ated in the TS dimer by the Asn177->Asp mutation. Results: A heterodim eric form of TS, containing one wild type and one Asn177->Asp active s ite, was created to test for negative cooperativity. Substrate inactiv ation still occurred, even with the mutation present at only one activ e site. Conclusions: Inactivation of TS(Asn177->Asp) by deoxyuridylate is not due to negative cooperativity created by the mutation. The 'ar tificial isozyme' method we have developed for purifying heterodimers away from the progenitor homodimers is generally applicable to other h etero-oligomeric proteins.