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Title: 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 (M/sub r/ 39,000) from Salmonella typhimurium are proteolyzed at homologous positions by ..cap alpha..-chymotrypsin, trypsin, and subtilisin to generate in all cases two nonoverlapping polypeptides of M/sub r/ 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-(/sup 14/C)-D-alanine, 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, NaB/sup 3/H/sub 4/-reduced DadB holoenzyme is resistant to ..cap 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.
Authors:
;
Publication Date:
OSTI Identifier:
5357602
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; (United States); Journal Volume: 26:25
Research Org:
Massachusetts Institute of Technology, Cambridge
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ISOMERASES; PROTEOLYSIS; POLYPEPTIDES; AMINO ACID SEQUENCE; ALANINES; CARBON 14 COMPOUNDS; DICHROISM; SALMONELLA TYPHIMURIUM; AMINO ACIDS; BACTERIA; CARBOXYLIC ACIDS; CHEMICAL REACTIONS; DECOMPOSITION; ENZYMES; LABELLED COMPOUNDS; MICROORGANISMS; MOLECULAR STRUCTURE; ORGANIC ACIDS; ORGANIC COMPOUNDS; PEPTIDES; PROTEINS; SALMONELLA 550201* -- Biochemistry-- Tracer Techniques