Mutations at the interdomain hinge region of the DadB alanine racemase: effects of length and conformational constraint of the linker sequence on catalytic efficiency.
The pentapeptide "hinge" region of the DadB alanine racemase links two structural domains of the protein [Galakatos, N. G., & 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.
Galakatos
N G
NG
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Walsh
C T
CT
D000411:Alanine Racemase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000591:Amino Acid Isomerases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D002942:Circular Dichroism; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000032:analysis; D010957:Plasmids; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013698:Templates, Genetic
D004269:DNA, Bacterial; D009841:Oligonucleotides; D000591:Amino Acid Isomerases; D000411:Alanine Racemase