Ligand-induced conformational changes of thymidylate synthase detected by limited proteolysis.
Limited tryptic proteolysis was used to investigate conformational changes of thymidylate synthase from Lactobacillus casei induced by ligand binding. Most of the identified sites of proteolysis were between R72 and R178, a region that includes a large loop containing residues 90-139 that is absent in thymidylate synthase from most other sources. Hydrolysis at both ends of this region was affected by the presence of dUMP. With dUMP, the preference of initial hydrolysis at the N-terminus of this region was switched from R78 to R72, and hydrolysis at R178 was retarded; the latter effect may be primarily a consequence of steric hinderance since R178 is involved in binding the phosphate moiety of dUMP. Orthophosphate had an effect similar to that of dUMP, not only in retarding hydrolysis at the phosphate binding site (R178) but also in retarding hydrolysis at R78 in favor of R72. Alkylation of the catalytically essential sulfhydryl group of thymidylate synthase by iodoacetamide also resulted in R72 being favored over R78 as a site of initial proteolysis. Its effect on hydrolysis at R178 was, as expected, less than that of dUMP or phosphate. These results indicate that dUMP binding induces conformational changes in thymidylate synthase. Phosphate binding and sulfhydryl alkylation also induce conformational changes similar to those resulting from dUMP binding. While the similarity of the proteolytic behavior of thymidylate synthase in the presence of dUMP or phosphate agrees with the report by Finer-Moore et al.(ABSTRACT TRUNCATED AT 250 WORDS)
Mohsen
A W
AW
Department of Animal and Dairy Sciences, Auburn University, Alabama 36849.
Aull
J L
JL
Payne
D M
DM
Daron
H H
HH
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003856:Deoxyuracil Nucleotides / Q000494:pharmacology; D006868:Hydrolysis; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D008969:Molecular Sequence Data; D010710:Phosphates / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D013940:Thymidylate Synthase / Q000737:chemistry*
D003856:Deoxyuracil Nucleotides; D010710:Phosphates; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase