Cellular energy metabolism
Studies have been carried out on adenylate kinase which is an important enzyme in determining the concentrations of the adenine nucleotides. An efficient method has been developed to clone mutant adenylate kinase genes in E. coli. Site-specific mutagenesis of the wild type gene also has been used to obtain forms of adenylate kinase with altered amino acids. The wild type and mutant forms of adenylate kinase have been overexpressed and large quantities were readily isolated. The kinetic and fluorescence properties of the different forms of adenylate kinase were characterized. This has led to a new model for the location of the AMP and ATP bindings sites on the enzyme and a proposal for the mechanism of substrate inhibition. Crystals of the wild type enzyme were obtained that diffract to at least 2.3 {angstrom} resolution. Experiments were also initiated to determine the function of adenylate kinase in vivo. In one set of experiments, E. coli strains with mutations in adenylate kinase showed large changes in cellular nucleotides after reaching the stationary phase in a low phosphate medium. This was caused by selective proteolytic degradation of the mutant adenylate kinase caused by phosphate starvation.
- Research Organization:
- Illinois Univ., Urbana, IL (United States). Dept. of Biochemistry
- Sponsoring Organization:
- USDOE; USDOE, Washington, DC (United States)
- DOE Contract Number:
- FG02-87ER13710
- OSTI ID:
- 5714213
- Report Number(s):
- DOE/ER/13710-T1; ON: DE92009045
- Country of Publication:
- United States
- Language:
- English
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PHOSPHOTRANSFERASES
ENZYME ACTIVITY
CLONING
DNA SEQUENCING
ESCHERICHIA COLI
GENES
NUCLEOTIDES
PLASMIDS
PROGRESS REPORT
BACTERIA
CELL CONSTITUENTS
DOCUMENT TYPES
ENZYMES
MICROORGANISMS
ORGANIC COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
PROTEINS
STRUCTURAL CHEMICAL ANALYSIS
TRANSFERASES
550400* - Genetics
550200 - Biochemistry