title: The effect of ligand binding on the proteolytic pattern of methylmalonate semialdehyde dehydrogenase.
abstract: Native rat liver methylmalonate semialdehyde dehydrogenase was proteolyzed by lysylendopeptidase C, chymotrypsin, and trypsin to generate different cleavage fragments of molecular masses: 50, 8, 55, 44, 39, 53, 45, and 40 kDa. A proteolytic cleavage map of MMSDH was constructed based on sequencing data and a comparison of appearance and degradation rates of the different protein fragments as shown by SDS-PAGE. NAD+ was highly effective as a protector against proteolysis in both the N-terminal and the C-terminal parts of the intact enzyme. NADH did not efficiently protect the intact enzyme; however, it stabilized proteolytic fragment L50 from further degradation. This suggests that the NAD(+)-binding domain is not destroyed by cleavage of the N-terminal part of MMSDH. CoA had no effect on the proteolytic cleavage patterns of MMSDH. However, CoA esters reduced the protective effect of NAD+ with an order of effectiveness of acetyl-CoA greater than propionyl-CoA greater than butyryl-CoA. p-Nitrophenyl acetate, substrate for esterase activity by the enzyme, partially prevented the protective effect of NAD+ against proteolysis. These results suggest that S-acylation of the enzyme prevents a stabilizing conformational change induced in MMSDH by NAD+ binding.
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122.
authors.forename: N Y
authors.initials: NY
authors.lastname: Kedishvili
authors.forename: K M
authors.initials: KM
authors.lastname: Popov
authors.forename: R A
authors.initials: RA
authors.lastname: Harris
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D066298:In Vitro Techniques; D008024:Ligands; D008099:Liver / Q000201:enzymology; D050646:Methylmalonate-Semialdehyde Dehydrogenase (Acylating); D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010447:Peptide Hydrolases; D051381:Rats
chemical_list: D008024:Ligands; D010446:Peptide Fragments; D009243:NAD; D000445:Aldehyde Oxidoreductases; D050646:Methylmalonate-Semialdehyde Dehydrogenase (Acylating); D010447:Peptide Hydrolases