Biomaterial Database

Site-directed mutagenesis of a nucleotide-binding domain in HSV-1 thymidine kinase: effects on catalytic activity. 1988

Q Y Liu, and W C Summers

Department of Molecular Biophysics and Biochemistry, Yale University, School of Medicine, New Haven, Connecticut 06510.

The thymidine kinase encoded by herpes simplex virus type 1 contains an amino acid sequence homologous to a consensus sequence related to the ATP-binding site in many proteins. We have used site-directed mutagenesis to investigate the importance of the five highly conserved amino acids within this segment. When any one of the three glycines was changed to valine the corresponding mutant enzyme was inactive. The mutation of lysine 63 to isoleucine destroyed the enzymatic activity. When threonine 64 was changed to alanine the mutant enzyme lost its activity. However, when this threonine was changed to serine the enzyme was still active but with different apparent Michaelis constants (Km) for thymidine and ATP. The wild-type thymidine kinase has apparent Km's of 0.5 and 20 microM for thymidine and ATP, respectively, while the mutant enzyme displayed Km's of 2.3 and 60 microM for thymidine and ATP. These results indicate that this homologous segment is essential for the function of the thymidine kinase and is involved in the substrate binding domain of the enzyme.

UI	MeSH Term	Description	Entries
D008239	Lysine	An essential amino acid. It is often added to animal feed.	Enisyl,L-Lysine,Lysine Acetate,Lysine Hydrochloride,Acetate, Lysine,L Lysine
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
D009154	Mutation	Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations.	Mutations
D004789	Enzyme Activation	Conversion of an inactive form of an enzyme to one possessing metabolic activity. It includes 1, activation by ions (activators); 2, activation by cofactors (coenzymes); and 3, conversion of an enzyme precursor (proenzyme or zymogen) to an active enzyme.	Activation, Enzyme,Activations, Enzyme,Enzyme Activations
D000255	Adenosine Triphosphate	An adenine nucleotide containing three phosphate groups esterified to the sugar moiety. In addition to its crucial roles in metabolism adenosine triphosphate is a neurotransmitter.	ATP,Adenosine Triphosphate, Calcium Salt,Adenosine Triphosphate, Chromium Salt,Adenosine Triphosphate, Magnesium Salt,Adenosine Triphosphate, Manganese Salt,Adenylpyrophosphate,CaATP,CrATP,Manganese Adenosine Triphosphate,MgATP,MnATP,ATP-MgCl2,Adenosine Triphosphate, Chromium Ammonium Salt,Adenosine Triphosphate, Magnesium Chloride,Atriphos,Chromium Adenosine Triphosphate,Cr(H2O)4 ATP,Magnesium Adenosine Triphosphate,Striadyne,ATP MgCl2
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
D013912	Threonine	An essential amino acid occurring naturally in the L-form, which is the active form. It is found in eggs, milk, gelatin, and other proteins.	L-Threonine,L Threonine
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