JBC, Vol. 250, Issue 13, 4864-4868, Jul, 1975
Interaction of deoxyuridylate with thymidylate synthetase
R. P. Leary, N. Beaudette and R. L. Kisliuk
The reaction of deoxyuridylate with Lactobacillus casei thymidylate
synthetase (methylenetetrahydrofolate: deoxyuridylate C-methyltransferase)
in the absence of folate or thiols can be followed by changes in the
circular dichroic spectra at 267 nm. These changes at the maximum
absorption wavelength of the pyrimidine suggest that on binding the
interaction of the pyrimidine ring and the deoxyribose moiety is altered.
The binding curve follows the form expected for single site binding. There
was no change in the ultraviolet absorption spectra. The dissociation
constant of the deoxyuridylate enzyme complex was calculated to be 4 x
10-minus 7 M. Iodoacetamide also reacts stoichiometrically with one of the
two subunits of thymidylate synthetase and inactivates the enzyme. This
reaction is blocked by the presence of deoxyuridylate or
chloromercuribenzoate. Complement fixation and immunodiffusion studies gave
no indication that iodoacetamide treatment caused conformational changes or
breakdown of the enzyme into subunits. Treatment with 0.1 M mercaptoethanol
restored activity to one-half the value obtained with native enzyme,
suggesting that mercaptoethanol enabled the unreacted subunit to become
functional.
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