Specific proteolysis of native alanine racemases from Salmonella typhimurium: identification of the cleavage site and characterization of the clipped two-domain proteins.
Native DadB and Alr alanine racemases (Mr 39,000) from Salmonella typhimurium are proteolyzed at homologous positions by alpha-chymotrypsin, trypsin, and subtilisin to generate in all cases two nonoverlapping polypeptides of Mr 28,000 and 11,000. Under nondenaturing conditions, chymotryptic digest results in an associated form of the two fragments which possesses 3% of the original catalytic activity, incorporates 0.76 equiv of the mechanism-based inactivator beta-chloro-[14C]-D-alanine [Badet, B., Roise, D., & Walsh, C. T. (1984) Biochemistry 23, 5188], and exhibits a UV circular dichroism profile identical with that of native enzyme. Protein sequence analysis of the denatured chymotryptic fragments indicates the presence of a tetrapeptide interdomain hinge (DadB, residues 254-257; Alr, residues 256-259) that is attacked at both ends during proteolysis. Under the previously employed digest conditions, NaB3H4-reduced DadB holoenzyme is resistant to alpha-chymotrypsin and trypsin and is labile only toward subtilisin. These data suggest that the hinge structure is essential for a catalytically efficient enzyme species and is sensitive to active site geometry. The sequence at the hinge region is also conserved in alanine racemases from Gram-positive bacteria.
Galakatos
N G
NG
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Walsh
C T
CT
D000411:Alanine Racemase / Q000378:metabolism*; D000591:Amino Acid Isomerases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000201:enzymology*; D012689:Sequence Homology, Nucleic Acid
D010446:Peptide Fragments; D010447:Peptide Hydrolases; D000591:Amino Acid Isomerases; D000411:Alanine Racemase