Arginine residues involved in binding of tetrahydrofolate to sheep liver serine hydroxymethyltransferase.
The arginine residue(s) necessary for tetrahydrofolate binding to sheep liver serine hydroxymethyltransferase were located by phenylglyoxal modification. The incorporation of [7-14C]phenylglyoxal indicated that 2 arginine residues were modified per subunit of the enzyme and the modification of these residues was prevented by tetrahydrofolate. In order to locate the sites of phenylglyoxal modification, the enzyme was reacted in the presence and absence of tetrahydrofolate using unlabeled and radioactive phenylglyoxal, respectively. The labeled phenylglyoxal-treated enzyme was digested with trypsin, and the radiolabeled peptides were purified by high-performance liquid chromatography on reversed-phase columns. Sequencing the tryptic peptides indicated that Arg-269 and Arg-462 were the sites of phenylglyoxal modification. Neither a spectrally discernible 495-nm intermediate (characteristic of the native enzyme when substrates are added) nor its enhancement by the addition of tetrahydrofolate, was observed with the phenylglyoxal-modified enzyme. There was no enhancement of the rate of the exchange of the alpha-proton of glycine upon addition of tetrahydrofolate to the modified enzyme as was observed with the native enzyme. These results demonstrate the requirement of specific arginine residues for the interaction of tetrahydrofolate with sheep liver serine hydroxymethyltransferase.
Usha
R
R
Department of Biochemistry, Indian Institute of Science, Bangalore.
Savithri
H S
HS
Rao
N A
NA
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D012696:Glycine Hydroxymethyltransferase / Q000378:metabolism*; D006898:Hydroxylamines / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010658:Phenylglyoxal / Q000378:metabolism; D011817:Rabbits; D012663:Semicarbazides / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D012756:Sheep; D013763:Tetrahydrofolates / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D006898:Hydroxylamines; D012663:Semicarbazides; D013763:Tetrahydrofolates; C030371:5,6,7,8-tetrahydrofolic acid; C005151:thiosemicarbazide; D001120:Arginine; C005214:methoxyamine; D012696:Glycine Hydroxymethyltransferase; D014357:Trypsin; D010658:Phenylglyoxal