Biochemistry 1991, 30, 4078-408 1
4078
Alkylation of an Active-Site Cysteinyl Residue during Substrate-Dependent Inactivation of Escherichia coli S-Adenosylmethionine Decarboxylaset Emilio Diaz* and David L. Anton* Central Research and Development Department, E. I . du Pont de Nemours and Company, Inc., Experimental Station, P.O.Box 80328, Wilmington, Delaware 19880-0328 Received September 12, 1990; Revised Manuscript Received December 26, 1990
S-Adenosylmethionine decarboxylase from Escherichia coli is a member of a small class of enzymes that uses a pyruvoyl prosthetic group. The pyruvoyl group is proposed to form a Schiff base with the substrate and then act as an electron sink facilitating decarboxylation. We have previously shown that once every 6000-7000 turnovers the enzyme undergoes an inactivation that results in a transaminated pyruvoyl group and the formation of an acrolein-like species from the methionine moiety. The acrolein then covalently alkylates the enzyme [Anton, D. L., & Kutny, R. (1987) Biochemistry 26, 64441. After reduction of the alkylated enzyme with NaBH,, a tryptic peptide with the sequence Ala-Asp-Ile-Glu-Val-Ser-Thr-[S-(3hydroxypropy1)Cysl-Gly-Val-Ile-Ser-Pro-Leu-Lys was isolated. This corresponds to acrolein alkylation of a cysteine residue in the second tryptic peptide from the NH2 terminal of the a-subunit [Anton, D. L., & Kutny, R. (1987) J . Biol. Chem. 262,2817-28221. The modified residue derived is from Cys-140 of the proenzyme [Tabor, C. W., & Tabor, H. (1987) J . Biol. Chem. 262, 16037-160401 and lies in the only sequence conserved between rat liver and E . coli S-adenosylmethionine decarboxylase [Pajunen et al. (1988) J . Biol. Chem. 263, 17040-170491. We suggest that the alkylated Cys residue could have a role in the catalytic mechanism. ABSTRACT:
S-Adenosylmethionine (AdoMet)' decarboxylase (EC 4.1.1 S O ) is a member of a small group of enzymes known to contain covalently bound pyruvoyl prosthetic groups, including histidine decarboxylase from several Gram-positive bacteria (Recsei & Snell, I984), aspartate a-decarboxylase from Escherichia coli (Williamson & Brown, 1979), phosphatidylserine decarboxylase from E . coli (Satre & Kennedy, 1978), proline reductase from Clostridia (Hcdgins & Abeles, 1967), and 4'-phosphopatothenoylcysteine decarboxylase from E. coli (Yang & Abeles, 1987). The product, decarboxylated AdoMet, is the source of the aminopropyl groups needed for the biosynthesis of the polyamines spermidine and spermine. The pyruvoyl group is thought to act analogously to pyridoxal phosphate cofactor, which is more commonly found in amino acid decarboxylases, by forming a Schiff base with the amino group of the substrate and then serving as an electron sink to facilitate the decarboxylation. We have previously shown that AdoMet decarboxylase from E. coli undergoes substrate-dependent inactivation that involves transamination of the pyruvoyl group with the substrate. This may occur after the normal decarboxylation reaction by incorrect protonation of the enolate intermediate (Scheme I) (Anton & Kutny, 1987a). This mechanism is analogous to the inactivation of pyridoxal-phosphate-dependent decarboxylases (O'Leary & Herreid, 1978). During inactivation of AdoMet decarboxylase there is an additional reaction involving the p elimination of methylthioadenosine to generate acrolein or an acrolein-like species, which stoichiometrically alkylates the enzyme (Scheme 11) (Anton & Kutny, 1987a). In this paper we report the 'Contribution No. 5491 from Central Research and Development, E. I . du Pont de Nemours and Co. A preliminary presentation of part of this work was presented at the American Society for Cell Biology and The American Society for Biochemistry and Molecular Biology Meeting, Jan 30, 1989, San Francisco, CA. Dedicated to Professor Robert H. Abclcs in honor of his 65th birthday. *Present address: Department of Chemistry, University of Puerto Rico, Mayaguez, PR 00708.
Scheme I 0
CHI
n 11
H - 0- C- CH- CHzCH2-
