In vivo mechanism-based inactivation of S-adenosylmethionine decarboxylases from Escherichia coli, Salmonella typhimurium, and Saccharomyces cerevisiae.
S-adenosylmethionine decarboxylase (AdoMetDC), a key enzyme in the biosynthesis of spermidine and spermine, is first synthesized as a proenzyme, which is cleaved posttranslationally to form alpha and beta subunits. The alpha subunit contains a covalently bound pyruvoyl group derived from serine that is essential for activity. With the use of an Escherichia coli overexpression system, we have purified AdoMetDCs encoded by the E. coli, Saccharomyces cerevisiae, and Salmonella typhimurium genes. Unexpectedly we found by mass spectrometry that these enzymes had been modified posttranslationally in vivo by a mechanism-based "suicide" inactivation. A large percentage of the alpha subunit of each enzyme had been modified in vivo to give peaks with masses m/z = 57 +/- 1 and m/z = 75 +/- 1 daltons higher than the parent peak. AdoMetDC activity decreased markedly during overexpression concurrently with the increase of the additional peaks for the alpha subunit. Sequencing of a tryptic fragment by tandem mass spectrometry showed that Cys-140 was modified with a +75 +/- 1 adduct, which is probably derived from the reaction product. Comparable modification of the alpha subunit was also observed in in vitro experiments after incubation with the substrate or with the reaction product, which is consistent with the in vitro alkylation of E. coli AdoMetDC reported by Diaz and Anton [Diaz, E. & Anton, D. L. (1991) Biochemistry 30, 4078-4081].
Li
Y F
YF
Laboratory of Biochemistry and Genetics, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Hess
S
S
Pannell
L K
LK
White Tabor
C
C
Tabor
H
H
D012437:Adenosylmethionine Decarboxylase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D002853:Chromatography, Liquid / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007544:Isopropyl Thiogalactoside; D013058:Mass Spectrometry / Q000379:methods; D011499:Protein Processing, Post-Translational; D012436:S-Adenosylmethionine / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D007544:Isopropyl Thiogalactoside; D012436:S-Adenosylmethionine; D012437:Adenosylmethionine Decarboxylase