AFFINITY PURIFICATION OF 5-METHYLTHIOADENOSINE KINASE AND 5-METHYLTHIORIBOSE S-ADENOSYLHOMOCYSTEIN NUCLEOSIDASE FROM KLEBSIELLA-PNEUMONIAE/

Two enzymes in the methionine salvage pathway, 5-methylthioribose kina se (MTR kinase) and 5'-methylthioadenosine/ S-adenosylhomocysteine nuc leosidase (MTA/SAH nucleosidase) were purified from Klebsiella pneumon iae. Chromatography using a novel inophenyl)thioadenosine/5-(paminophe nyl)thioribose affinity matrix allowed the binding and selective eluti on of each of the enzymes in pure form. The molecular mass, substrate kinetics and N-terminal amino acid sequences were characterized for ea ch of the enzymes. Purified MTR kinase exhibits an apparent molecular mass of 46-50 kDa by SDS/PAGE and S200HR chromatography, and has a K-m for MTR of 12.2 mu M. Homogeneous MTA/SAH nucleosidase displays a mol ecular mass of 26.5 kDa by SDS/PAGE, and a K-m for MTA of 8.7 mu M. Co mparisons of the N-terminal sequences obtained for each of the enzymes with protein-sequence databases failed to reveal any significant sequ ence similarities to known proteins. However, the amino acid sequence obtained for the nucleosidase did share a high degree of sequence simi larity 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.