An anomaly in the active site region of thymidylate synthase.
A putative thyA gene from Escherichia coli was cloned into a high expression vector and the thymidylate synthase produced was purified to homogeneity. Comparison of the monomer molecular weight of this protein with that of authentic E. coli thymidylate synthase revealed the two to differ, suggesting that they were derived from different sources. This was confirmed by Ochterlony immunodiffusion analysis, which revealed that while the unknown thymidylate synthase formed a precipitin band with guinea pig antibody to the putative E. coli synthase, pure E. coli TS did not. In addition, the specific enzyme activity of the purified unknown thymidylate synthase was about 4-fold higher than that of the pure authentic enzyme. Sequence analysis of the active site peptide revealed that the amino acid linked to the carboxyl end of the active site cysteine was valine. The only instance where this has been found in the 11 thymidylate sequences reported so far is in the thyP3 sequence of the Bacillus subtilis phage-3T. In all the other cases, a histidine has been found in this position. Amino end group sequence analysis of the unknown synthase for about 30 residues confirmed the close identity of this protein to that of the B. subtilis phage thymidylate synthase. To determine whether the replacement of the active site histidine with a valine enhances the activity of the resulting thymidylate synthase, we affected this change in T4-phage thymidylate synthase by site-directed mutagenesis and found that instead of an increase in activity there was an 80 percent decrease.
Maley
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Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201-0509.
Maley
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D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D006639:Histidine; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D013604:T-Phages / Q000201:enzymology; D013940:Thymidylate Synthase / Q000235:genetics / Q000378:metabolism*; D014633:Valine
D011994:Recombinant Proteins; D006639:Histidine; D013940:Thymidylate Synthase; D014633:Valine