Probing the infra-structure of thymidylate synthase and deoxycytidylate deaminase.

Methods are described for preparing and structurally analyzing two enzymes involved in the formation of dTMP, deoxycytidylate deaminase and thymidylate synthase. In the latter case, it has been possible through the use of recombinant DNA techniques with an amplification plasmid to obtain sufficient amounts of the E. coli and T4-phage synthases to complete the entire sequence of both enzymes by employing a combination of protein and DNA sequencing methods. A comparative analysis of the L. casei and E. coli synthases has revealed a 62% conservation of sequences but an even greater homology in their hydrophobic active site regions (82%), which are primarily hydrophobic in nature. The homology between these enzymes becomes apparent by deleting a 51 amino acid segment (residues 89-139) from the L. casei synthase, which accounts for the difference in size between these enzymes. Methods for obtaining the binding sites of both substrates are described, one being the activation of the carboxyls of folate with a water soluble carbodiimide and the other, the activation of dUMP by ultraviolet light. The DNA and protein sequence of the T4-phage synthase has recently been clarified by us and is in preparation. Of great interest is the finding by Purohit and Mathews (42), based on our sequence data for the synthase, that the gene segment for the carboxyl terminal end of dihydrofolate reductase overlaps with the amino end of the gene for thymidylate synthase. The complete amino acid sequence of T2-phage deoxycytidylate deaminase has been elucidated by conventional protein sequencing methods. The binding characteristics of this enzyme for its positive allosteric effectors and substrates, as determined by equilibrium dialysis, are consistent with the cooperative nature of its kinetic responses. Consistent with these findings was the demonstration that each of the enzyme's six subunits bound an equivalent amount of substrate or allosteric modifier. Similarly the deaminase showed a marked negative change in ellipticity at 280 nm in response to increasing concentrations of dCTP, changes which could be reversed by dTTP. From the information on the enzyme's primary sequence, it should be possible to define the substrate and allosteric binding regions within the deaminase with the appropriately activated compounds. A start in this direction has been initiated by the finding that dTTP is rapidly and apparently covalently fixed to the amino terminal cyanogen bromide peptide of the enzyme in the presence of ultraviolet light.