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Herpes thymidine kinase mutants with altered catalytic efficiencies obtained by random sequence selection
K M Munir1, D C French, D K Dube
1Joseph Gottstein Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle 98195.
Protein Engineering
|January 1, 1994
Summary
Researchers created Herpes simplex virus type 1 thymidine kinase mutants to study enzyme activity. Some mutants showed enhanced or significantly reduced thymidine kinase function, demonstrating a range of catalytic properties from random sequence substitutions.
Area of Science:
- Molecular Biology
- Enzymology
- Virology
Background:
- Herpes simplex virus type 1 (HSV-1) thymidine kinase is a crucial enzyme in viral DNA replication.
- Understanding the structure-function relationship of HSV-1 thymidine kinase is vital for antiviral drug development.
- Previous studies have focused on rational design for enzyme modification.
Purpose of the Study:
- To generate and characterize a library of HSV-1 thymidine kinase mutants with altered substrate binding sites.
- To investigate the catalytic properties of these mutants in relation to thymidine phosphorylation.
- To explore the feasibility of generating diverse enzyme activities through random mutagenesis and selection.
Main Methods:
- Generated 190 active HSV-1 thymidine kinase mutants by random nucleotide sequence substitutions in the thymidine binding site.
- Assessed mutant enzyme activity by measuring the ability of Escherichia coli harboring the mutants to form colonies in the presence of varying thymidine concentrations.
- Determined in vivo and in vitro kinetic parameters, including [methyl-3H]thymidine uptake, incorporation into acid-insoluble material, and enzyme kcat.
Main Results:
- Identified mutants with enhanced thymidine kinase activity, exhibiting increased thymidine phosphorylation, higher [methyl-3H]thymidine uptake (42%), and greater incorporation into acid-insoluble material (2.4-fold).
- One mutant enzyme showed a 5-fold increase in kcat compared to the wild type.
- Other mutants displayed significantly reduced catalytic activity, with one exhibiting a kcat that was 10(-4) times that of the wild type enzyme.
Conclusions:
- Random sequence substitutions in the HSV-1 thymidine kinase gene can generate a wide spectrum of mutant enzymes with diverse catalytic properties.
- This approach allows for the selection of enzymes with enhanced or diminished activity without prior rational protein design.
- The study highlights the potential of random mutagenesis and selection for enzyme engineering and functional studies.