Biochemical and immunological characterization of deoxyhypusine synthase purified from the yeast Saccharomyces carlsbergensis.
Deoxyhypusine synthase catalyzes the NAD+-dependent formation of deoxyhypusine in the eIF-5A precursor protein by transferring the 4-aminobutyl moiety of spermidine. This enzyme has recently been shown to be essential for cell viability and growth of yeast [Sasaki, K., Abid, M.R., and Miyazaki, M. (1996) FEBS Lett. 384, 151 154]. We have purified and characterized the enzyme from the yeast Saccharomyces carlsbergensis. The yeast and recombinant enzymes had a specific activity of 1.21 to 1.26 pmol per min per pmol of protein, and recognized both the eIF-5A precursor proteins almost equally as judged from their similar K(m) and V(max) values. Size exclusion chromatography and SDS-PAGE indicated that the active form of the enzyme is a homotetramer consisting of 43-kDa subunits. The enzyme showed a strict specificity for its substrates, NAD+, spermidine and eIF-5A precursor protein. Among all the substrates tested, only NAD+ showed a protective effect against heat inactivation of the enzyme suggesting that NAD+ initiates some conformational change in the enzyme. NADH exhibited a strong non-competitive inhibition (product inhibition). Unexpectedly, FAD, FMN, and riboflavin showed a moderate competitive inhibition. The competitive inhibition by diamines was maximal with compounds resembling spermidine in carbon chain length. 1,3-Diaminopropane inhibited the enzyme strongly in a competitive manner (product inhibition). On the other hand, putrescine did not inhibit the enzyme or act as a substrate. A polyclonal antibody raised against the yeast recombinant enzyme specifically inhibited deoxyhypusine synthase activity. The cross-reactivity (by Western blotting) of this antibody with the crude extracts varied depending on the source, indicating species specificity.
Abid
M R
MR
Department of Molecular Biology, School of Science, Nagoya University, Chikusa-ku.
Sasaki
K
K
Titani
K
K
Miyazaki
M
M
D000595:Amino Acid Sequence; D000818:Animals; D003429:Cross Reactions; D003959:Diamines / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008322:Mammals / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009491:Neurospora / Q000201:enzymology; D009711:Nucleotides / Q000494:pharmacology; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000502:physiology*; D011073:Polyamines / Q000494:pharmacology; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012440:Saccharomyces / Q000737:chemistry / Q000201:enzymology*; D017421:Sequence Analysis; D013045:Species Specificity; D013696:Temperature
D003959:Diamines; D004791:Enzyme Inhibitors; D009711:Nucleotides; D011073:Polyamines; D011994:Recombinant Proteins; D000587:Oxidoreductases Acting on CH-NH Group Donors; C063507:deoxyhypusine synthase