Purification and properties of 7, 8-diaminopelargonic acid aminotransferase.
The enzyme 7, 8-diaminopelargonic acid aminotransferase utilizes S-adenosyl-L-methionine to transaminate the biotin precurson 7-keto-8-aminopelargonic acid and form the next intermediate in the pathway, 7, 8-diaminopelargonic acid. The enzyme has been purified nearly 1000-fold from an extract of a regulatory mutant of Escherichia coli which is derepressed for the enzymes of the biotin operon. The extract was treated with protamine sulfate, ammonium sulfate, and subjected to acid and heat treatments. Subsequently, the enzyme was chromatographed on columns of DEAE-cellulose, phosphocellulose, hydroxylapatite, and two Sephadex G-100. The resulting purified preparation was judged 86% homogeneous by the scanning of of a stained disc gel. The enzymatic activity was associated with the major band in gels run at two different gel concentrations and two different pH values. The cofactor, pyridoxal phosphate, can be resolved from the enzyme in the presence of phosphate buffer after incubation with the amino donor, S-adenosyl-L-methionine. A molecular weight estimation of 94,000 plus or minus 10, 000 has been obtained by gel filtration and sucrose gradient sedimentation studies. Gel electrophoresis in the presence of sodium dodecyl sulfate, shows a single subunit with a molecular weight of 47, 000 plus or minus 3, 000 indicating a dimeric enzyme. A neutral compound was detected in the acidified reaction mixture which was derived from the methionine moiety of S-adenosyl-L-methionine and was present in amounts equivalent to the 7, 8-diaminopelargonic acid produced in the reaction mixture. It is suggested that the keto product of the reaction, i.e. S-adenosyl-2-oxo-4-methylthiobutyric acid, may decompose nonenzymatically under the conditions of the reaction to form 5'-methylthioadenosine and the neutral compound, 2-oxo-3-butenoic acid.
D000599:Amino Acids, Diamino / Q000494:pharmacology; D001681:Biological Assay; D001710:Biotin / Q000096:biosynthesis*; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004355:Drug Stability; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D006358:Hot Temperature; D006882:Hydroxyapatites; D007651:Keto Acids; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011479:Protamines; D012436:S-Adenosylmethionine; D013997:Time Factors; D000637:Transaminases / Q000302:isolation & purification* / Q000378:metabolism
D000599:Amino Acids, Diamino; D006882:Hydroxyapatites; D007651:Keto Acids; D046911:Macromolecular Substances; D011479:Protamines; D001710:Biotin; D012436:S-Adenosylmethionine; D000637:Transaminases