INTERACTION OF THYMIDYLATE SYNTHASE WITH PYRIDOXAL 5'-PHOSPHATE AS STUDIED BY UV VISIBLE DIFFERENCE SPECTROSCOPY AND MOLECULAR MODELING/

Pyridoxal 5'-phosphate (PLP) is an effective inhibitor of Lactobacillu s casei thymidylate synthase (TS), competitive with respect to the nuc leotide substrate dUMP (Chen et al., 1989). The UV/vis difference spec tra of TS-PLP complexes show lambda(max) at 328 nm due to the specific interaction between Cys 198 of TS and PLP to form a thiohemiacetal, a nd lambda(min) at 388 nm due to depletion of free PLP. At high concent rations of PLP a new absorbance at 430 nm forms due to nonspecific Sch iff base formation between PLP and lysine residues of the enzyme. Usin g spectral titration at 328 nm, the binding constant of the specific T S-PLP complex was determined to be 0.5 muM, and the stoichiometry was 2 mol of PLP/mol of TS dimer. The 328-nm absorbance of the TS-PLP comp lex can be competitively and completely eliminated by addition of dUMP or dTMP; this serves as a convenient binding assay for molecules whic h bind to the active site of TS. Analogs of PLP which do not contain t he phosphate or the aldehyde moieties of PLP bound poorly to the enzym e, thus demonstrating the importance of these functional groups for bi nding. When treated with PLP, C244T TS, which contains the active site Cys 198 as the sole cysteine residue, showed the same properties as t he wild-type enzyme. Treatment of the C198A and C198S mutants with PLP did not produce the absorbance at 328 nm assigned to thiohemiacetal f ormation. Molecular modeling studies showed that when the phosphate of PLP is docked into the phosphate binding site of TS, the aldehyde car bon of PLP is perfectly positioned to form a thiohemiacetal with the c atalytic thiol of Cys 198. Side chains of amino acid residues of the p rotein which are close to or may interact with PLP were tentatively as signed.