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. 1996

T M Rechtin, and M E Black, and R R Drake
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, 72205, USA.

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.

UI MeSH Term Description Entries
D008666 Metalloendopeptidases ENDOPEPTIDASES which use a metal such as ZINC in the catalytic mechanism. Metallo-Endoproteinases,Metalloendopeptidase
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D010449 Peptide Mapping Analysis of PEPTIDES that are generated from the digestion or fragmentation of a protein or mixture of PROTEINS, by ELECTROPHORESIS; CHROMATOGRAPHY; or MASS SPECTROMETRY. The resulting peptide fingerprints are analyzed for a variety of purposes including the identification of the proteins in a sample, GENETIC POLYMORPHISMS, patterns of gene expression, and patterns diagnostic for diseases. Fingerprints, Peptide,Peptide Fingerprinting,Protein Fingerprinting,Fingerprints, Protein,Fingerprint, Peptide,Fingerprint, Protein,Fingerprinting, Peptide,Fingerprinting, Protein,Mapping, Peptide,Peptide Fingerprint,Peptide Fingerprints,Protein Fingerprint,Protein Fingerprints
D000345 Affinity Labels Analogs of those substrates or compounds which bind naturally at the active sites of proteins, enzymes, antibodies, steroids, or physiological receptors. These analogs form a stable covalent bond at the binding site, thereby acting as inhibitors of the proteins or steroids. Affinity Labeling Reagents,Labeling Reagents, Affinity,Labels, Affinity,Reagents, Affinity Labeling
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D001665 Binding Sites The parts of a macromolecule that directly participate in its specific combination with another molecule. Combining Site,Binding Site,Combining Sites,Site, Binding,Site, Combining,Sites, Binding,Sites, Combining
D013936 Thymidine A nucleoside in which THYMINE is linked to DEOXYRIBOSE. 2'-Deoxythymidine,Deoxythymidine,2' Deoxythymidine
D013937 Thymidine Kinase An enzyme that catalyzes the conversion of ATP and thymidine to ADP and thymidine 5'-phosphate. Deoxyuridine can also act as an acceptor and dGTP as a donor. (From Enzyme Nomenclature, 1992) EC 2.7.1.21. Deoxythymidine Kinase,Deoxypyrimidine Kinase,Kinase, Deoxypyrimidine,Kinase, Deoxythymidine,Kinase, Thymidine
D018259 Herpesvirus 1, Human The type species of SIMPLEXVIRUS causing most forms of non-genital herpes simplex in humans. Primary infection occurs mainly in infants and young children and then the virus becomes latent in the dorsal root ganglion. It then is periodically reactivated throughout life causing mostly benign conditions. HSV-1,Herpes Simplex Virus 1,HHV-1,Herpes Simplex Virus Type 1,Herpesvirus 1 (alpha), Human,Human Herpesvirus 1

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