Affinity purification of 5-methylthioadenosine kinase and 5-methylthioribose/ S-adenosylhomocysteine nucleosidase from Klebsiella pneumoniae

Kenneth A. Cornell, R. W. Winter, Paula A. Tower, Michael Riscoe

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

Two enzymes in the methionine salvage pathway, 5-methylthioribose kinase (MTR kinase) and 5′-methylthioadenosine/ S-adenosylhomocysteine nucleosidase (MTA/SAH nucleosidase) were purified from Klebsiella pneumoniae. Chromatography using a novel 5′-(p-aminophenyl)thioadenosine/5-(p-aminophenyl)thioribose affinity matrix allowed the binding and selective elution of each of the enzymes in pure form. The molecular mass, substrate kinetics and N-terminal amino acid sequences were characterized for each of the enzymes. Purified MTR kinase exhibits an apparent molecular mass of 46-50 kDa by SDS/PAGE and S200HR chromatography, and has a Km for MTR of 12.2 μM. Homogeneous MTA/SAH nucleosidase displays a molecular mass of 26.5 kDa by SDS/PAGE, and a Km for MTA of 8.7 μM. Comparisons of the N-terminal sequences obtained for each of the enzymes with protein-sequence databases failed to reveal any significant sequence similarities to known proteins. However, the amino acid sequence obtained for the nucleosidase did share a high degree of sequence similarity with the putative translation product of an open reading frame in Escherichia coli, thus providing a tentative identification of this gene as encoding an MTA/SAH nucleosidase.

Original languageEnglish (US)
Pages (from-to)285-290
Number of pages6
JournalBiochemical Journal
Volume317
Issue number1
StatePublished - Jul 1 1996

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adenosylhomocysteine nucleosidase
Klebsiella pneumoniae
Purification
Molecular mass
Enzymes
Chromatography
Pemetrexed
Polyacrylamide Gel Electrophoresis
Amino Acid Sequence
N-Glycosyl Hydrolases
Amino Acids
Salvaging
Protein Databases
Methionine
Escherichia coli
Open Reading Frames
Proteins
Phosphotransferases
Genes
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Affinity purification of 5-methylthioadenosine kinase and 5-methylthioribose/ S-adenosylhomocysteine nucleosidase from Klebsiella pneumoniae. / Cornell, Kenneth A.; Winter, R. W.; Tower, Paula A.; Riscoe, Michael.

In: Biochemical Journal, Vol. 317, No. 1, 01.07.1996, p. 285-290.

Research output: Contribution to journalArticle

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