Substitution of glutamine for glutamic acid-58 in Escherichia coli thymidylate synthase results in pronounced decreases in catalytic activity and ligand binding.

The recent determination of the crystal structure of Escherichia coli thymidylate synthase (TS) [Matthews et al. (1989) J. Mol. Biol. 205, 449-454] has implicated the glutamic acid residue at position 58 in a mechanistic role which could involve the interaction of its gamma-carboxyl side chain with the nucleotide substrate and/or the folate cofactor. The site-specific mutagenesis of Glu-58 to Gln-58 in E. coli TS provided the opportunity to explore its functional role in activity and binding. When profiled by the spectrophotometric and tritium release assays, the 370- and 760-fold decreases, respectively, in kcat and the elevated Km values for the Gln-58 mutant enzyme indicated a significant involvement of Glu-58 in substrate binding and turnover. The apparent dissociation constant for the covalent FdUMP-enzyme binary complex was 30 microM, which is 5-fold higher than that found for the wild-type enzyme, while the inhibitory ternary complex apparent dissociation constants for FdUMP and CH2H4folate for the Gln-58 enzyme were 10- and 60-fold higher, respectively, than those for the wild-type enzyme under saturating conditions. The extent of covalent FdUMP binding to the Gln-58 enzyme was reduced from 1.5 to 0.7 per dimer in the inhibitory ternary complex but only from 0.7 to 0.5 per dimer in the binary complex of the Gln-58 enzyme. The usual 2.1-fold enhancement of FdUMP binding to wild-type TS in the presence of CH2H4folate was not observed for the Gln-58 enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of selenomethionyl thymidylate synthase from Escherichia coli: comparison with the wild-type enzyme.

Replacement of methionine (Met) residues by selenomethionine (SeMet) was recently shown to facilitate the crystallographic analysis of protein structure through the application of multi-wavelength anomalous diffraction techniques [Yang et al. (1990) Science (Washington, D.C.) 249, 1398-1405]. The availability of SeMet-containing proteins provides an excellent opportunity to evaluate the effects of the complete replacement of Met by SeMet. We chose to compare the properties of selenomethionyl thymidylate synthase isolated from Escherichia coli DL41 (a methionine auxotroph) and wild-type (wt) enzyme obtained from E. coli Rue10. An improved purification procedure for thymidylate synthase was developed which permitted the isolation of 25 mg of pure protein from 2 g of E. coli in 90% yield in no more than 8 h. The pure wt and SeMet enzymes exhibited specific activities 40% higher than published values. Thermal stability studies at 30 degrees C in degassed buffer showed that the SeMet enzyme (t1/2 67 h) was 8-fold less stable than wt enzyme (t1/2 557 h). The half-lives for the latter enzymes in nondegassed buffers at 30 degrees C were decreased by 2-fold, thus indicating the sensitivity of the enzyme to dissolved oxygen. Both enzymes exhibited essentially the same kinetic and binding properties, including Km(dUMP) (1.2 x 10(-6) M), specificity constant (1.6 x 10(6) s-1 M-1), and Kd for 5-fluorodeoxyuridylate binding (1.2 nM) in covalent inhibitory ternary complexes. In addition, X-ray crystallographic analysis by difference Fourier synthesis showed there was no significant difference in conformation between the SeMet enzyme and the wt enzyme.

Effect of oral contraceptives and pregnancy on erythrocyte deformability and surface charge.

Erythrocyte deformability and surface charge were studied in normal premenopausal women, oral contraceptive users, and pregnant women. The increased incidence of thrombosis in women taking oral contraceptives could not be explained by decreased erythrocyte deformability or surface charge. However, the decreased erythrocyte deformability of late pregnancy may relate to thrombosis during this period and to increased hemolysis in patients with certain hemoglobinopathies.

PIP: Erythrocyte membrane deformability is a factor in blood viscosity. Progesterone and synthetic progestational steroids interact with the ery throcyte membrane and may be related to the decreased deformability. In women, reduction in net negative surface charge during pregnancy has bee n reported. This might overcome normal especially with a reduced rate of blood flow. These factors were investigated in blood taken from patients in a Family Planning Clinic and an Obstetrical Clinic. Patients taking oral contraceptives had used them for more than 2 months. Techniques employed are described. Erythrocyte deformability was unchanged in the contraceptive users. In pregnant women a difference in filtration time was observed, indicating decreased deformity of the erythrocytes. Net negative surface charge, as measured by electrophoretic mobility, was unchanged in oral contraceptive users and in pregnant women. The increased incidence of thrombosis in women taking oral contraceptives could not be explained by decreased erythrocyte deformability or surface charge. However, decreased deformability of erythrocytes in late pregnancy may relate to thrombosis during this period, and to increased hemolysis in patients with some hemoglobinopathies.