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 impo rtant pharmacological target of antiviral nucleoside drugs and it uniq uely possesses both a thymidine kinase and a thymidylate kinase activi ty. The structural relationship between these two activities is addres sed in this study using a combination of active-site directed photoaff inity analogs, proteases, and tricine-SDS-polyacrylamide gel electroph oresis, For analysis of the thymidylate binding site, the thymidylate analog [P-32]5-azido-dUMP was specifically photocrosslinked to the act ive site of HSV-1 TR. Because the amino acid sequence of HSV-1 TK is k nown, endoprotease Lys-C, V8 protease, trypsin, or chymotrypsin was us ed to generate a proteolytic map of photoincorporated peptides by sepa ration on high-resolution tricine-SDS-polyacrylamide gels. Analysis of the resulting peptides indicated that the photoprobe was localized to one region comprising amino acids Ile(112)-Tyr(132). 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 t echnique 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. (C) 1996 Academic Press, Inc.