Biochemical and immunological characterization of deoxyhypusine synthase purified from the yeast Saccharomyces carlsbergensis.

Abstract:

: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.

journal_name

J Biochem

journal_title

Journal of biochemistry

authors

Abid MR,Sasaki K,Titani K,Miyazaki M

doi

10.1093/oxfordjournals.jbchem.a021652

subject

Has Abstract

pub_date

1997-04-01 00:00:00

pages

769-78

issue

4

eissn

0021-924X

issn

1756-2651

journal_volume

121

pub_type

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