Mass spectrometric identification of K210 essential for rat malonyl-CoA decarboxylase catalysis.
Proteomic technology provides useful tools to detect protein modification sites in vivo and in vitro. In this work, we applied proteomics to identify an essential amino acid residue involved in Malonyl-CoA Decarboxylase (MCD) catalysis. A reaction with acetic anhydride and MCD, under mild conditions without acetyl CoA as a substrate, resulted in the acetylation of six lysyl residues, K210, K58, K167, K316, K388, and K444. When acetyl CoA was added to the reaction, K210 was protected from acetylation, indicating a potential role for this residue in catalysis. In addition, K210 was the only lysyl residue, out of six, that was not endogenously acetylated. Because K210, K308, and K388 are conserved across species, they were site-specifically mutated to methionine which is size-wise similar to lysine but not protonated. The K308M and K388M MCD mutants retained 60% of their enzyme activities, whereas the K210M mutant was completely inactive. These results strongly suggest that K210 is an essential residue in rat MCD catalysis and is a likely proton donor to the alpha carbon of malonyl-CoA. Therapeutic inhibition of MCD may be a viable approach to treating various clinical pathologies associated with defective fatty acid metabolism.
Nam
Hyung Wook
HW
Department of Biochemistry, College of Science, Protein Network Research Center, Yonsei University, Seoul, Korea 120-749.
Lee
Gha Young
GY
Kim
Yu Sam
YS
D000089:Acetic Anhydrides / Q000378:metabolism; D000105:Acetyl Coenzyme A / Q000378:metabolism*; D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008715:Methionine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011499:Protein Processing, Post-Translational / Q000235:genetics / Q000502:physiology*; D040901:Proteomics; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization
D000089:Acetic Anhydrides; C031800:acetic anhydride; D000105:Acetyl Coenzyme A; D008715:Methionine; D002262:Carboxy-Lyases; C021532:malonyl-CoA decarboxylase; D008239:Lysine