Specific proteolysis of native alanine racemases from Salmonella typhimurium: identification of the cleavage site and characterization of the clipped two-domain proteins

Journal Article · Tue Dec 15 04:00:00 EST 1987 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00399a066· OSTI ID:5357602

Galakatos, N G; Walsh, C T

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.

• Research Organization: Massachusetts Institute of Technology, Cambridge
• OSTI ID: 5357602
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:25; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALANINES
AMINO ACID SEQUENCE
AMINO ACIDS
BACTERIA
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
DECOMPOSITION
DICHROISM
ENZYMES
ISOMERASES
LABELLED COMPOUNDS
MICROORGANISMS
MOLECULAR STRUCTURE
ORGANIC ACIDS
ORGANIC COMPOUNDS
PEPTIDES
POLYPEPTIDES
PROTEINS
PROTEOLYSIS
SALMONELLA
SALMONELLA TYPHIMURIUM