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Title:Gene cloning, adjustable expression and site-directed mutagenesis of Escherichia coli adenylate kinase
Author(s):Liang, Peng
Doctoral Committee Chair(s):Glaser, Michael
Department / Program:Biology, Molecular
Discipline:Biology, Molecular
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biology, Molecular
Abstract:The gene encoding the Escherichia coli adenylate kinase has been cloned by phage Mu-mediated transposition. Subcloning of the adk$\sp{+}$ gene into pUC13 and pBR322 allowed high-level expression and a simple purification scheme for the enzyme. An optimized system was developed for the in vivo transfer of a mutant gene from the Escherichia coli chromosome to a plasmid carrying the wild type allele. A 15% frequency of homogenotization was obtained during cloning of a adk gene that codes for a temperature-sensitive adenylate kinase with a point mutation from leu107 to gln. Measurement of the mass action ratio of the adenylate kinase reaction indicated that adenylate kinase may be rate limiting in growing cells and that mutant adenylate kinases were selectively degraded by phosphate starvation. However, adjustable expression of adenylate kinase from 30% below the level of the mutant to 50-fold above the wild-type level did not have any effect on the adenine nucleotide concentrations. Site-directed mutagenesis was used to investigate the location of the ATP and AMP binding sites on adenylate kinase and the mechanism of substrate inhibition. Two amino acids, phe86 and tyr133, which were found to be in close contact with the multisubstrate analogue Ap$\sb5$A in previous X-ray studies were independently changed to tryptophan and other amino acids. Both enzyme kinetics and tryptophan fluorescence data suggest that the phe86 is located in the active site of the enzyme and primarily involved in the binding of AMP, while tyr133 plays more a structural role rather than being directly involved in nucleotide binding. The results also suggest that AMP substrate inhibition is caused by the formation of an abortive complex at high AMP concentrations that prevent the release of the product. A model is proposed where phe86 is critically involved in AMP binding, leu107 is near the ATP binding site, and tyr133 is not directly involved in nucleotide binding.
Issue Date:1990
Rights Information:Copyright 1990 Liang, Peng
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9026253
OCLC Identifier:(UMI)AAI9026253

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