The conserved Asn 229 of thymidylate synthase (TS) forms a cyclic hydrogen bond network with the 3-NH and 4-O of the nucleotide substrate 2'-deoxyuridine 5'-monophosphate (dUMP). Asn 229 is not essential for substrate binding or catalysis [Liu, L., gr Santi, D, V, (1993) Proc. Natl. Acad. Sci, U.S.A, 90, 8604-8608] but is a major determinant in substrate specificity [Liu, L., & Santi, D. V. (1993) Biochemistry 32, 9263-9267], 3-Methyl-dUMP (3-MedUMP) is neither a substrate nor an inhibitor of wild type TS but is converted to 3-methyl 2'-deoxythymidine 5'-monophosphate by many TS Asn 229 mutants. Some of the Asn 229 mutants (N229C, -I, -M, -A, and -V) have k(cat) values for 3-MedUMP methylation which are up to about 20% of that for wild type TS-catalyzed methylation of JUMP, and some mutants (N229C and -A) catalyze methylation of 3-MedUMP more efficiently than that of dUMP. Mutants with hydrophobic side chains tended to be mon active in catalysis of methylation of 3-MedUMP than those with hydrophilic side chains. The ability of 3-MedUMP to serve as a substrate for Asn 229 mutants shows that the active form of dUMP involves the neutral pyrimidine base and that ionization of the 3-NH group does not occur in the course of catalysis, In contrast to the negligible binding of 3-MedUMP to wild type TS, both 3-MedUMP and dUMP showed similar K-m values with the Asn 229 mutants, suggesting similar binding affinities to the mutants. The X-ray crystal structure of the TS N229C-3-MedUMP complex showed that the side chain of Cys 229 was rotated away from the pyrimidine ring to allow placement of a water molecule and the 3-methyl group of 3-MedUMP ill the active site. Our results suggest that the inability of 3-MedUMP to undergo methylation by wild type TS is due to its inability to bind to the enzyme, which in turn is simply a result of steric Interference of the 3-methyl group with the side chain of Asn 229.
Attenzione! Scheda prodotto non ancora validata dall'Ateneo
|Anno di pubblicazione:||1996|
|Titolo:||Asparagine 229 mutants of thymidylate synthase catalyze the methylation of 3-methyl-2'-deoxyuridine 5'-monophosphate|
|Autori:||Costi MP; Liu L; FinerMoore JS; Stroud RM; Santi DV|
|Appare nelle tipologie:||Articolo su rivista|
File in questo prodotto:
I documenti presenti in Iris Unimore sono rilasciati con licenza Creative Commons Attribuzione - Non commerciale - Non opere derivate 3.0 Italia, salvo diversa indicazione.
In caso di violazione di copyright, contattare Supporto Iris