J. Biochem, 1997, Vol. 121, No. 4 769-778
© 1997 Japanese Biochemical Society
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Biochemical and Immunological Characterization of Deoxyhypusine Synthase Purified from the Yeast Saccharomyces carlsbergensis
*Department of Molecular Biology, School of Science, Nagoya University Chikusa-ku, Nagoya 464–01
Institute for Comprehensive Medical Science, Fujita Health University Toyoake, Aichi 470–11
1To whom correspondence should be addressed. Tel: +81-052-789-2991, Fax: +81-052-789-3001 E-mail: j45933a{at}nucc.cc.nagoya-u.ac.jp
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 eEF-5A precursor proteins almost equally as judged from their similar Kmand Vmax 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 eD7-5A 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.
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