Purification of human S-adenosylmethionine decarboxylase expressed in Escherichia coli and use of this protein to investigate the mechanism of inhibition by the irreversible inhibitors, 5'-deoxy-5'-[(3-hydrazinopropyl)methylamino]adenosine and 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine.
Human S-adenosylmethionine decarboxylase (AdoMetDC) was expressed in high yield in Escherichia coli using the pIN-III(lppP-5) expression vector and purified to apparent homogeneity using affinity chromatography on methylglyoxal bis(guanylhydrazone)-Sepharose. The inactivation of the purified enzyme by 5'-deoxy-5'-[(3-hydrazinopropyl)methylamino]adenosine (MHZPA) was accompanied by an increase in absorbance at 260 nm of the large subunit. This increase was equivalent to the addition of 1 molecule of MHZPA. After digestion with the protease Lys-C, a peptide that contained the bound MHZPA was isolated and found to have the amino acid composition consistent with that expected from the amino terminus of the large subunit. These results indicate that MHZPA inactivates AdoMetDC by forming a hydrazone derivative at the pyruvate prosthetic group. Inactivation of AdoMetDC by 5'-([(Z)-4-amino-2-butenyl]methylamino]-5'-deoxyadenosine (AbeAdo) led to the appearance of a new peptide peak in the Lys-C protease digest. This peptide had the sequence ASMFVSK. This agrees with the expected sequence from the amino terminus, which is pyruvoyl-SMFVSK, with the exception that the pyruvate has been converted to alanine. Direct gas-phase sequencing of the large subunit of the enzyme also indicated the presence of alanine at the amino terminus after inactivation with AbeAdo. These results indicate that this inhibitor leads to transamination of the pyruvate prosthetic group. Since the pyruvate is covalently linked to the protein, its replacement by alanine leads to an irreversible inactivation of AdoMetDC.
Shantz
L M
LM
Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey 17033.
Stanley
B A
BA
Secrist
J A
JA
Pegg
A E
AE
D012437:Adenosylmethionine Decarboxylase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D003839:Deoxyadenosines / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification
D003062:Codon; D003839:Deoxyadenosines; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C057410:5'-deoxy-5'-(N-methyl-N-(3-hydrazinopropyl)amino)adenosine; C065859:MDL 73811; D012437:Adenosylmethionine Decarboxylase