Proteolytic mapping of the thymidine/thymidylate binding site of herpes simplex virus type 1 thymidine kinase: a general photoaffinity labeling method for identifying active-site peptides.
The herpes simplex virus type 1 thymidine kinase (HSV-1 TK) is an important pharmacological target of antiviral nucleoside drugs and it uniquely possesses both a thymidine kinase and a thymidylate kinase activity. The structural relationship between these two activities is addressed in this study using a combination of active-site directed photoaffinity analogs, proteases, and tricine-SDS-polyacrylamide gel electrophoresis. For analysis of the thymidylate binding site, the thymidylate analog [32P]5-azido-dUMP was specifically photocrosslinked to the active site of HSV-1 TK. Because the amino acid sequence of HSV-1 TK is known, endoprotease Lys-C, V8 protease, trypsin, or chymotrypsin was used to generate a proteolytic map of photoincorporated peptides by separation on high-resolution tricine-SDS-polyacrylamide gels. Analysis of the resulting peptides indicated that the photoprobe was localized to one region comprising amino acids Ile112-Tyr132. Photolabeling of this region indicates that the thymine base of thymidine and TMP bind at one shared site in HSV-1 TK. In addition, the results reported in this study demonstrate that photolabeling with azidonucleotides can be used to identify photolabeled peptides by proteolytic mapping. This technique bypasses the problems of peptide purification and sequencing and yields rapid results when the primary amino acid structure of the protein of interest is known.
Rechtin
T M
TM
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, 72205, USA.
Black
M E
ME
Drake
R R
RR
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D008666:Metalloendopeptidases / Q000494:pharmacology; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D013936:Thymidine / Q000378:metabolism*; D013937:Thymidine Kinase / Q000378:metabolism*
D000345:Affinity Labels; D013937:Thymidine Kinase; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D013936:Thymidine