Mutations at the interdomain hinge region of the DadB alanine racemase: effects of length and conformational constraint of the linker sequence on catalytic efficiency
- 1 October 1989
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 28 (20), 8167-8174
- https://doi.org/10.1021/bi00446a031
Abstract
The pentapeptide "hinge" region of the DadB alanine racemase links two structural domains of the protein [Galakatos, N. G., and Walsh, C. T. (1987) Biochemistry 26, 8475]. The presence of substrate markedly reduces the rate of hinge-specific proteolysis of this racemase and induces a conformational change observed by circular dichroism. To evaluate the possible contribution of the proteolytically sensitive hinge residues (-Y253GGGY257-) on catalytic efficiency, site-directed mutations were generated to probe the effects of size and conformational rigidity of that region. A bacterial overproducing system for the dadB gene was constructed that expresses the enzyme as 4.5% of total soluble protein. On this construct, a four-part ligation allowed the engineering of two unique and proximal restriction sites required for cassette mutagenesis at the hinge region. For two of the eight mutants generated, expressed protein could not be detected (deletion of -GGGY-; termination codon at position 255). Deletion of one or two of the three Gly residues had no effect on catalytic efficiency. Insertion of a fourth Gly resulted in a 5-fold drop in Vmax/Km. For G254P, G255P, and G256P, Vmax/Km was 60%, 126%, and 26% of the native enzyme, respectively. In all cases, the Km''s remained essentially constant, suggesting that the hinge region is not involved in substrate binding. The rate of hinge-specific proteolysis of the mutants was faster than that of wild-type DadB except for the G255P protein for which it was equivalent.This publication has 18 references indexed in Scilit:
- Conformational changes in aspartate aminotransferase. Effect of active site ligands on peptide hydrogen-deuterium exchange.Journal of Biological Chemistry, 1985
- Chemical synthesis of a gene encoding the human complement fragment C5a and its expression in Escherichia coli.Proceedings of the National Academy of Sciences, 1985
- Identification of the dadX gene coding for the predominant isozyme of alanine racemase in Escherichia coli K12Molecular Genetics and Genomics, 1985
- Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sitesGene, 1985
- Catabolic alanine racemase from Salmonella typhimurium: DNA sequence, enzyme purification, and characterizationBiochemistry, 1984
- Mechanisms of domain closure in proteinsJournal of Molecular Biology, 1984
- Two alanine racemase genes in Salmonella typhimurium that differ in structure and functionJournal of Bacteriology, 1983
- Mitochondrial aspartate aminotransferase 27/32-410. Partially active enzyme derivative produced by limited proteolytic cleavage of native enzyme.Journal of Biological Chemistry, 1980
- A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye BindingAnalytical Biochemistry, 1976
- PROTEIN MEASUREMENT WITH THE FOLIN PHENOL REAGENTJournal of Biological Chemistry, 1951