Asn177 in Escherichia coli thymidylate synthase is a major determinant of pyrimidine specificity.
The substrate preference of recombinant Escherichia coli thymidylate synthase (TS) has been altered from 2'-deoxyuridylate (dUMP) to 2'-deoxycytidylate (dCMP) by site-directed mutagenesis of the codon for Asn177, which was changed to aspartic acid. The side-chain amide of Asn177 forms hydrogen bonds with O4 and N3 of dUMP bound to the crystalline enzyme [Montfort, W. R., Perry, K. M., Fauman, E. B., Finer-Moore, J. S., Maley, G. F., Hardy, L., Maley, F. & Stroud, R. M. (1990) Biochemistry 29, 6964-6977]. This Asn is invariant in all natural sequences for TS known. The values of kcat for the mutant enzyme, TS(N177D), with dCMP and dUMP are, respectively, 0.09 and 0.002 times the value of kcat of wild-type TS with dUMP as substrate. TS(N177D) turns over dCMP at 35 times its rate of dUMP turnover, whereas wild-type TS turns over dCMP at < 10(-5) of its rate of dUMP turnover. Thus Asn177 is a major determinant of the pyrimidine nucleotide specificity of TS. The mutant enzyme, like wild-type TS, forms a covalent complex with 5-fluoro-dUMP in the presence of 5,10-methylenetetrahydrofolate. TS(N177D) also has a newly acquired ability to be transiently inactivated by dUMP. This time-dependent inactivation requires the presence of methylenetetrahydrofolate and may be due to the accumulation of the enzyme in the form of a catalytic intermediate. The likely mechanistic basis for discrimination by TS between dUMP and dCMP is their differing requirements for charge stabilization during covalent catalysis.
Hardy
L W
LW
Department of Pharmacology, University of Massachusetts Medical Center, Worcester 01605.
Nalivaika
E
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D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011743:Pyrimidines / Q000378:metabolism*; D013379:Substrate Specificity; D013940:Thymidylate Synthase / Q000737:chemistry* / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D011743:Pyrimidines; D001224:Aspartic Acid; D001216:Asparagine; D013940:Thymidylate Synthase