(Received for publication, January 17, 1997, and in revised form, March 28, 1997)
From the Oral and Pharyngeal Cancer Branch, NIDR, National Institutes of Health, Bethesda, Maryland 20892-4340
Deoxyhypusine
(N-(4-aminobutyl)lysine) is the key
intermediate in the posttranslational synthesis of the unique amino
acid, hypusine
(N
-(4-amino-2-hydroxybutyl)lysine).
Deoxyhypusine synthase catalyzes the formation of deoxyhypusine by
conjugation of the butylamine moiety of spermidine to the
-amino
group of one specific lysine residue of the eukaryotic translation
initiation factor 5A (eIF-5A) precursor protein. However, in the
absence of the eIF-5A precursor, catalysis involves only the
NAD-dependent cleavage of spermidine to generate
1,3-diaminopropane and a putative 4-carbon amine intermediate that
gives rise to
1-pyrroline. We have obtained evidence for
a covalent enzyme-substrate intermediate that accumulates in the
absence of the eIF-5A precursor. Incubation of human recombinant enzyme
with [1,8-3H]spermidine and NAD, followed by reduction
with NaBH3CN, resulted in specific radiolabeling of the
enzyme. The radioactive component in the reduced enzyme intermediate
was identified as deoxyhypusine and was shown to occur at a single
locus. The fact that labeled deoxyhypusine was found after treatment
with a reducing agent suggests an intermediate with the butylamine
moiety derived from spermidine attached through an imine linkage to the
-amino group of a specific lysine residue of the enzyme. This
residue has been identified as lysine 329. Separate experiments showing
efficient transfer of labeled butylamine moiety from enzyme
intermediate to eIF-5A precursor strongly support a reaction mechanism
involving an imine intermediate.
The posttranslational formation of deoxyhypusine
(N-(4-aminobutyl)lysine) is catalyzed
by deoxyhypusine synthase and involves modification of a single lysine
residue (Lys50 in the human protein) of the
eIF-5A1 precursor (for reviews, see Refs.
1-3). It is the first step in the biosynthesis of
N
-(4-amino-2-hydroxybutyl)lysine
(hypusine), the unusual amino acid that characterizes mature eIF-5A (4,
5). This 17-kDa protein and the enzymes that catalyze its modification
are present in all eukaryotic cells. There is mounting evidence that
hypusine synthesis is vital for proliferation in mammalian cells (2, 6-8) and those of other eukaryotic species. Yeast cells are not viable
if the two genes for eIF-5A have been inactivated, whereas expression
of either one permits growth (9-12). Furthermore, it has been shown
very recently that the presence of an intact gene for deoxyhypusine
synthase (13)2 and expression of
deoxyhypusine synthase activity2 are essential for yeast
viability. Effective inhibitors of deoxyhypusine synthase have been
developed; these inhibitors cause inhibition of hypusine synthesis in
eIF-5A in cells and also have been shown to inhibit proliferation of
various mammalian cells (6, 14, 15). In view of the position of the
enzyme as the first step in the biosynthesis of hypusine, such
inhibitors could have an important place in therapy of
hyperproliferative conditions. A thorough understanding of the
mechanism and regulation of this enzyme is therefore of great
interest.
Deoxyhypusine synthase has been purified from rat testis (16), Neurospora crassa (17), and HeLa cells (18). The gene was identified in yeast (13, 18-20), and human and Neurospora cDNAs have been cloned (20-22). The recombinant enzymes, purified after expression of the human or yeast cDNA in Escherichia coli, are now available in essentially homogeneous form. The amino acid sequence of the enzyme is highly conserved; it appears that the enzyme from all species studied exists as a tetramer of identical subunits of 40-43 kDa (depending on the species) and that the cofactor requirements and catalytic properties of the enzyme from different species are similar (16, 19, 21).
Earlier studies with the purified enzyme from rat testis (16, 23), and
with the human and yeast recombinant enzymes (19, 21), have shown that
the complex reaction leading to the modification of the eIF-5A
precursor protein through deoxyhypusine synthesis is carried out by a
single enzyme. In a complete reaction mixture containing spermidine,
NAD, and eIF-5A precursor, deoxyhypusine synthase catalyzes an
NAD-dependent cleavage of spermidine to generate free
1,3-diaminopropane and an enzyme-bound 4-aminobutyl moiety, which
is subsequently transferred to a lysine of the eIF-5A precursor to form
the deoxyhypusine residue. However, if the eIF-5A precursor is absent
from the reaction mixture, deoxyhypusine synthase catalyzes only part
of this reaction, namely the cleavage of spermidine; in this case
1,3-diaminopropane and 1-pyrroline are observed as
products (16, 19, 21, 23). The role of dehydrogenation of spermidine
was established in experiments that showed the transfer of
3H from [5-3H]spermidine to NAD as a critical
initial step of the reaction (23). Based on these and other early
observations, which established the origin of the atoms of
deoxyhypusine (24-26), we proposed an overall scheme, similar to that
shown in Scheme 1. The proposed mechanism involved a dehydrospermidine
intermediate, probably tightly bound to the enzyme (23), and possibly
an enzyme-imine intermediate (3, 16). However, there was no tangible
evidence for the existence of a covalent enzyme-substrate intermediate. We speculated that if a transient intermediate with the 4-carbon amine
moiety from spermidine attached to the enzyme, e.g. through an imine linkage, is involved in the catalysis, it might be possible to
trap the unsaturated intermediate in a stable form by the use of a
reducing agent such as NaBH4. Following the pioneering
observations of Fischer et al. (28), numerous examples have
appeared in the literature of detection of unsaturated enzyme
intermediates by this method (29-35). The present results, summarized
in Scheme 1, indicate that such an unsaturated enzyme-substrate
intermediate derived from spermidine
(Enz-N=CH(CH2)3NH2 in Scheme 1) is
indeed formed by modification of a specific lysine residue, namely
Lys329 of human deoxyhypusine synthase. Furthermore, we
present evidence that this postulated intermediate can act as a
catalytic transfer intermediate in deoxyhypusine synthesis in
eIF-5A.
Materials
[1,8-3H]Spermidine·3HCl (15-26.5 Ci/mmol) was purchased from DuPont NEN; TLC plastic sheets with Silica gel 60 (without fluorescent indicator), 20 × 20 cm, thickness 0.2 mm (EM Science catalog no. 5748) were obtained from ACE Scientific Co., EN3HANCE from DuPont NEN. Precast polyacrylamide gels, sample buffer, Tricine running buffer, and colloidal Coomassie Blue stain were from Novex; NAD from Boehringer Mannheim; Coomassie Brilliant Blue R250 from AT Biochem; trypsin (TPCK-treated, 232 units/mg) and chymotrypsin (TLCK-treated, 49.0 units/mg) from Worthington; Chromatography columns Mono-Q® (HR 10/10 and 5/10) and Mono-P® HR 5/20, and matrices Butyl-Sepharose and Q-Sepharose from Pharmacia Biotech Inc., hydroxylapatite (dry powder, Lot 410043) from Calbiochem; NaBH4 and NaBH3CN from Aldrich. NaBH3CN was recrystallized by the method of Jentoft and Dearborn (35) and kept dessicated.
Methods
Peptide SynthesisThe synthetic peptides, Gly-deoxyhypusine
(Dhp)-Ile-Arg and Gly-Dhp-Glu-Leu-Arg, was prepared by a solid phase
procedure (36) with the use of
t-Boc-Arg(Tos)OCH2PAM resin (Applied Biosystems) and tert-butyloxycarbonyl -amino group protection. The
amino-protected deoxyhypusine derivative,
tert-butyloxycarbonyl-N6-p-nitrobenzyloxycarbonyl-N6-(4-p-nitrobenzyloxycarbonylaminobutyl)-(2S)-2,6-diaminohexanoic acid, was prepared as outlined (37).
t-Boc-Glu-
-benzyl ester was used in the synthesis of the
latter peptide. The peptides were partially deprotected and removed
from the resin with HF containing 10% m-cresol. Removal of
the p-nitrobenzyloxycarbonyl groups from the side chain of
the deoxyhypusine residue was then carried out by hydrogenation in 50%
aqueous methanol in the presence of palladium black catalyst. The
peptides were purified by chromatography on CM-Sephadex C-25
(NH4+ form, Pharmacia) using a linear
gradient of increasing strength of NH4OH. Examination of
acid hydrolysates of the peptides by thin layer chromatography showed,
in each case, approximately equal amounts of each of the composite
amino acids.
Purification of recombinant enzyme from E. coli B strain BL21 (DE23) expressing human deoxyhypusine synthase cDNA followed the published procedure (21) with modifications. Because the enzyme prepared as described earlier (21) that appeared homogeneous on SDS-PAGE in a Tris-glycine buffer system in fact contained a significant amount of a bacterial protein of nearly the same molecular mass, as became apparent upon SDS-PAGE in a Tricine buffer system, an additional step was taken. To separate the enzyme from the contaminating bacterial protein, chromatography was conducted on a butyl-Sepharose column. The sample after Mono-Q chromatography (16) was applied to this column equilibrated with 0.05 M Tris acetate, pH 7.5, containing 0.1 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 1 M (NH4)2SO4. Elution was made with an 80-ml linear gradient from equilibration buffer to the same buffer without (NH4)2SO4. The enzyme activity eluted just after the conductivity dropped to ~40% of the initial value. This preparation, estimated to be >95% pure (specific activity 900 units/µg, where 1 unit = formation of 1 pmol of deoxyhypusine/h) was used for the experiments reported in this study. The enzyme activity was assayed by the published procedure (16, 23).
Generation of an Enzyme-Substrate Intermediate and Its Reduction by NaBH3CNHighly purified human deoxyhypusine
synthase (1-3 µl, 2-6 µg) was added to mixtures of reactants
preheated at 37 °C in siliconized Eppendorf tubes to give,
typically, 20 µl containing 1 mM dithiothreitol, 5%
glycerol, 500 µM NAD, and 7.5 µM
[1,8-3H]spermidine (4 µCi) in 0.2 M
glycine-NaOH buffer, pH 9.5. After 2 min, or the specified time, at
37 °C, the tubes were transferred to an ice bath and
NaBH3CN was added (1 µl of 0.1 M in
H2O, freshly prepared from the recrystallized compound, for
a total of 3-4 portions at 10-min intervals) to a final concentration
of 15-20 mM. Specific details are given in the figure
legends. For SDS-PAGE, 25-100 µg of bovine serum albumin was added
to a portion of each reaction mixture and the proteins were
precipitated with 10% trichloroacetic acid. The precipitates were
dissolved in Tricine sample buffer containing mercaptoethanol, adjusted
to neutral pH with Tris base, placed in boiling water for 3-4 min, and
subjected to SDS-PAGE on a precast 10% (Novex) Tricine gel. The gel
was stained with Coomassie Blue, destained, soaked in water for 30 min
and in 1 M sodium salicylate for 60 min, and exposed to
Kodak X-Omat AR x-ray film at 70 °C. For ion-exchange
chromatographic analysis of deoxyhypusine and polyamines, another
portion of the enzyme-labeling reaction mixture was precipitated with
10% trichloroacetic acid in the presence of 500 µg of carrier BSA,
the precipitates washed three or four times with 10% trichloroacetic
acid containing 2 mM spermidine, and subjected to acid
hydrolysis in 6 N HCl at 105 °C for 16 h. The
hydrolysate and the trichloroacetic acid-soluble reaction products were
analyzed by an ion-exchange chromatography system described previously
(38) employing sodium citrate/NaCl buffers: Buffer A (0.6 N
Na+, pH 5.8), Buffer B (1.5 N Na+,
pH 5.55), Buffer C (3 N Na+, pH 5.55) for 5, 10, and 30 min, respectively. The identification of reaction products
was as reported previously (23).
For
sequence determination, labeled human deoxyhypusine synthase was
prepared as outlined above on a large scale, using 400 µg of enzyme
in a final volume of 2 ml; [1,8-3H]spermidine was diluted
with non-radioactive spermidine to a final concentration of 108 µM (400 µCi/2 ml). After reduction of the enzyme
intermediate, protein was precipitated with trichloroacetic acid
without addition of bovine serum albumin, and the precipitates were
washed thoroughly with 10% trichloroacetic acid containing 2 mM spermidine, followed by ice-cold acetone (two washes).
The protein was digested for 10 h at 37 °C in 100 mM ammonium bicarbonate, pH 8.1, containing 0.15 units of
trypsin (300 units/mg) and 0.1% reduced Triton (Sigma). The trypsin
level was brought to 0.3 units, and the digestion was continued for an
additional 14 h. Separation of peptides was performed on a 2 mm × 250 mm C-18 column (Vydac) in 0.1% trifluoroacetic acid in
HPLC water; a gradient was then applied over 90 min bringing the
elution buffer (90% acetonitrile, 10% HPLC water, 0.085%
trifluoroacetic acid) to 65% acetonitrile. A hydrophobic radiolabeled
peptide eluting as a single peak later in the gradient was identified
as peptide A by electrophoresis and TLC as described in the legend to
Fig. 5. Another radiolabeled peptide eluted from the C-18 early in the
gradient was identified as peptide B; after further purification on an
ion-exchange column, the radioactive peak fraction was desalted using a
Perkin-Elmer Applied Biosystems C-18 column, 1.0 mm × 50 mm, in
0.1% trifluoroacetic acid, brought to 50% acetonitrile:50% water
over 30 min. This peptide was subjected to 7 cycles of Edman automated
degradation to yield GXIR as a major sequence; no equivalent
amount of amino acid derivative was seen after the fourth cycle. The
tryptic digestion, separation of peptides, and sequence determination
by automated Edman degradation were carried out at the Michigan State
Macromolecular Structure Facility under the direction of Joseph F. Leykam, Department of Biochemistry, Michigan State University, East
Lansing, MI.
It
was observed previously that deoxyhypusine synthase acts to cleave
spermidine to 1,3-diaminopropane and a 4-carbon amine moiety both in
the presence and absence of the eIF-5A precursor, although the nature
of the 4-carbon product is different in the two cases. The butylamine
moiety becomes the extended side chain of deoxyhypusine in the eIF-5A
precursor protein in the former case and free
1-pyrroline in the latter (23). We reasoned that if a
common, transient intermediate is involved in formation of the two
4-carbon products, it would be more likely to accumulate in the absence of eIF-5A precursor, since in this case the spermidine cleavage reaction proceeds at a much slower rate. If, after the initial dehydrogenation of spermidine and cleavage of the N4-C5 bond, the
resulting butylamine moiety is covalently attached to enzyme through an
unsaturated linkage, it should be possible to produce a stable
derivative of the postulated intermediate by reduction of the reaction
mixture. We succeeded in demonstrating that, in the absence of the
normal acceptor of the butylamine moiety, the eIF-5A precursor,
radioactivity from [1,8-3H]spermidine was indeed retained
on the enzyme protein after brief incubation of deoxyhypusine synthase,
NAD and [1,8-3H]spermidine if the reaction mixture was
subsequently treated with either NaBH4 or
NaBH3CN. The labeling appeared to be more efficient with
NaBH3CN than NaBH4, as noted by others (34,
35). The results of a typical experiment are shown in Fig.
1. In the Coomassie Blue-stained gel (Fig.
1A), the single band of human deoxyhypusine synthase is
shown at ~41 kDa (indicated by the arrow), along with
several bands from bovine serum albumin, added to aid in the
trichloroacetic acid precipitation of protein before SDS-PAGE. The
fluorogram of the same gel (Fig. 1B) shows one band of
radioactivity, which is at the position of the enzyme
(arrow), only in the sample in which the enzyme was
incubated with NAD and [3H]spermidine, followed by
reduction with NaBH3CN (Fig. 1B, lane 5). No labeled product was detectable if enzyme or NAD was omitted from the reaction mixture (Fig. 1B, lanes 1-3)
or when reducing agent was not added (Fig. 1B, lanes
2 and 4).
Identification of Deoxyhypusine as the Product of Reduction of the Enzyme Intermediate
The radioactive component of the
3H-labeled enzyme band excised from a gel or of the
trichloroacetic acid precipitates of a reaction mixture (Fig.
2A) was identified as deoxyhypusine by ion
exchange chromatography after acid hydrolysis (see "Experimental Procedures"). A single peak of radioactivity, corresponding in position to that of deoxyhypusine, was observed (Fig. 2A,
). No detectable [3H]deoxyhypusine was found in those
samples where NAD was omitted (Fig. 2A,
), or where
NaBH3CN was not added (data not shown). In this experiment,
during the 2-min incubation, 21.1 pmol of deoxyhypusine/100 pmol of
enzyme monomer was formed. Analysis by ion-exchange chromatography of
the trichloroacetic acid-soluble radioactive products (Fig.
2B) formed from [1,8-3H]spermidine revealed
pyrrolidine (the reduction product of
1-pyrroline; Ref.
23) and 1,3-diaminopropane. Interestingly, the amount of pyrrolidine is
significantly less than the amount of 1,3-diaminopropane generated (7.3 versus 30.3 pmol, respectively); the difference can largely
be accounted for by the amount of the 4-aminobutyl moiety incorporated
into the enzyme and trapped as deoxyhypusine (21.1 pmol) (Fig.
2A). On the other hand, in a parallel reaction not treated
with NaBH3CN, the
1-pyrroline and
1,3-diaminopropane were found in nearly equal quantities (ratio of
0.8), as observed previously (23). This can be taken as evidence that
the enzyme intermediate
(Enz-N=CH(CH2)3NH2 in Scheme
1), if not trapped by NaBH3CN reduction,
gives rise to
1-pyrroline.
Time Course of Formation of Deoxyhypusine Synthase-Substrate Intermediate
The level of the enzyme intermediate, measured as
the amount of deoxyhypusine after reduction (Fig.
3A), reached a peak at ~2 min and appeared
to decrease slowly thereafter. A parallel increase in
1,3-diaminopropane occurred in the first 2 min (Fig. 3B).
However, in a second phase of slower product formation, the amount of
1,3-diaminopropane and 1-pyrroline (measured as
pyrrolidine, see previous section) continued to increase with time
(Fig. 3B). Reduction in the level of spermidine corresponded
to the increase in 1,3-diaminopropane. The sum of the amounts of
1-pyrroline and intermediate correlates generally with
the amounts of 1,3-diaminopropane, as discussed in the preceding
section. The fact that the enzyme-substrate intermediate did not
continue to accumulate, while the other products did, suggests that
intermediate formation is balanced by its degradation to
1-pyrroline and free enzyme.
Catalytic Competence of the Deoxyhypusine Synthase-Substrate Intermediate
The postulated enzyme-substrate intermediate should
be able to transfer its butylamine moiety to the specific lysine of the eIF-5A precursor if it is a true catalytic intermediate in the reaction. The intermediate, however, is not stable, i.e. it
breaks down spontaneously in the absence of eIF-5A precursor, as is
evident from the results in Fig. 3. Rather than attempting physical
isolation of this transient enzyme-substrate intermediate free from
[3H]spermidine, we chose to block its further formation
with the use of an inhibitor. 1-Amino,7-guanidinoheptane (GC7) by
competition effectively prevents the productive binding of spermidine
to the enzyme (11). At 100 µM this inhibitor totally
stopped intermediate formation, as shown in Fig.
4A (lane 5). That the labeled
butylamine moiety from the preformed intermediate can be transferred to
eIF-5A precursor, and that the transfer is not prevented by the
inhibitor is evident from the data of Fig. 4B. Deoxyhypusine
synthase was incubated for a short time with
[1,8-3H]spermidine and NAD to accumulate the enzyme
intermediate. Reduction followed by PAGE shows evidence for labeled
enzyme-intermediate formation (Fig. 4B, lane 1).
To this mixture, containing the enzyme intermediate,
1-amino-7-guanidinoheptane (100 µM) was added to stop
further formation of the intermediate. When human recombinant eIF-5A
precursor, ec-eIF-5A, was added after the inhibitor, radioactivity appeared within 1 min at the position of the ec-eIF-5A, both in reduced
(lanes 2-5) and unreduced (lanes 7-10) samples.
No label remained in the enzyme band in the reduced sample (lanes
2-5), demonstrating that complete transfer of the butylamine
moiety from the enzyme intermediate to the eIF-5A precursor had
occurred prior to reduction. In a control reaction, in which inhibitor was included from the onset (Fig. 4B, lane 6), no
labeling of either enzyme-intermediate or eIF-5A precursor was
observed, confirming that intermediate formation was blocked by the
inhibitor. The radioactive component of the modified ec-eIF-5A was
identified as deoxyhypusine in both reduced and unreduced samples,
showing a normal course of deoxyhypusine production from the
enzyme-substrate intermediate, i.e. transfer of the
butylamine moiety followed by enzyme-catalyzed reduction.
Identification of Lysine 329 as the Enzyme Residue Involved in Intermediate Formation
Human deoxyhypusine synthase labeled with
[3H]spermidine was digested with trypsin. A peptide map
of the digest displayed a single radiolabeled peptide (Peptide
A, Fig. 5A). Chymotrypsin treatment of
the tryptic digest containing peptide A provided a distinctly different
labeled peptide (Peptide B, Fig. 5B). Prolonged tryptic digestion (40 h) or the use of high levels of trypsin alone
also produced peptide B, probably due to a chymotryptic activity
recognized to be residual in trypsin even after TPCK treatment, that is
enhanced by autodigestion (41). Peptide B is clearly more basic than
peptide A, judging by its migration in electrophoresis (Fig. 5), and
probably very hydrophilic, as it migrated very little in the
chromatography mixture used (Fig. 5). In separate experiments, peptide
B was shown to be much smaller than peptide A (2.5 kDa
versus ~3.5-6 kDa) by SDS-PAGE on a 16% Tricine gel
(data not shown). It seems evident from these results that peptide B is
derived from peptide A by a chymotryptic cleavage, with the radiolabel
residing in peptide B.
Fig. 6A shows the pattern of radioactivity
obtained upon Edman degradation of labeled peptide B. The large
majority of radioactivity appeared at the second Edman cycle.
Examination of the deduced amino acid sequence of human deoxyhypusine
synthase (21) revealed two locations where a conserved lysine is the
second residue after a tryptic or chymotryptic cleavage site and where
the expected peptide would have the characteristics of basicity,
hydrophilicity, and small size expected from the results described
above. These regions of the sequence, residues 155-159 and 328-331,
along with the flanking 3 residues in each case, are shown in Fig.
6B. The sequence, GXIR, where X is an
unidentified residue, was found as a major sequence in a parallel Edman
degradation of partially purified peptide B; it is evident that the
unidentified residue, X, corresponds to the position of
radioactivity shown in Fig. 6A, and to the position of
Lys329 in the unmodified human enzyme (Fig. 6B).
Chymotryptic-like cleavage at Trp327 of the larger peptide
A would explain the origin of peptide B. Synthetic peptides
corresponding to each of these sequences with deoxyhypusine in place of
lysine were prepared (see "Experimental Procedures") and subjected
to electrophoresis and chromatography, in two solvent systems, along
with radiolabeled Peptide B from the digest (above). The synthetic
peptide Gly-Dhp-Ile-Arg exactly co-migrated with the radioactivity in
peptide B, whereas the other peptide, Gly-Dhp-Glu-Leu-Arg, did not
(Fig. 6C). Thus, the deoxyhypusine found in the modified
enzyme after reduction of the enzyme-substrate intermediate must result
from modification of Lys329 of the human enzyme.
The present study was undertaken to extend our knowledge of the deoxyhypusine synthase reaction. The expectation that an enzyme-substrate intermediate might form with the 4-carbon amine moiety from spermidine covalently attached to deoxyhypusine synthase through an unsaturated linkage was based on the earlier observation that the dehydrogenation of spermidine between N4 and C5, as shown by the transfer of 3H from [5-3H]spermidine to NAD (23), is a critical obligatory step preceding spermidine cleavage and deoxyhypusine synthesis (see Scheme 1). However, it was not known whether the transfer of the butylamine moiety to eIF-5A precursor occurs directly or through a covalent enzyme intermediate. In one possible scenario, the eIF-5A precursor protein could bind to the enzyme in close proximity to spermidine and transfer of the butylamine moiety could proceed directly to the specific lysine residue of the substrate protein. Thus, one function of the enzyme would be to bring the two substrates into juxtaposition for the transfer. In addition, the enzyme might play a more active role by accepting the butylamine moiety from spermidine to form an enzyme-substrate intermediate and then delivering it to the protein substrate. The current data provide compelling evidence for such a covalent intermediate function for the enzyme.
Identification of labeled deoxyhypusine as a component of modified deoxyhypusine synthase after reduction of enzyme-substrate intermediate defines a lysine residue of the enzyme itself as an acceptor of the 4-aminobutyl moiety from spermidine. That the labeled deoxyhypusine residue on the modified enzyme was generated from an unsaturated intermediate by introduction of hydrogen was confirmed with the use of [3H]NaBH4. Radiolabeled deoxyhypusine was produced when an incubation mixture of the enzyme, NAD, and unlabeled spermidine were reduced with [3H]NaBH4 (data not shown).
Examination of amino acid sequences of the labeled peptides derived from the reduced enzyme-substrate intermediate (Fig. 6) revealed that modification occurred at one specific lysine (Lys329) of human deoxyhypusine synthase. No evidence for involvement of any other residue was obtained. The specificity of the transfer exclusively to this lysine suggests that this is not a random event, but rather is a specific transfer near or at the active site of the enzyme, that takes place immediately following the dehydrogenation of spermidine. However, it was not clear initially whether this reflected an obligatory intermediate in the pathway leading to the production of deoxyhypusine in the eIF-5A intermediate form, eIF-5A(Dhp) (Scheme 1), or an abortive side-product resulting from the absence of the normal acceptor of the butylamine moiety, the eIF-5A precursor. Strong evidence in favor of the former is derived from the observed capability of the enzyme-substrate intermediate to complete a cycle in the deoxyhypusine synthesis when eIF-5A precursor protein is added (Fig. 4).
While these studies were in progress, we conducted independent site-directed mutagenesis studies designed to assess the requisite role of several conserved lysine residues in catalysis. A human mutant enzyme with substitution of Ala for Lys329 did not form an enzyme intermediate that could be trapped by NaBH3CN and had only a trace of deoxyhypusine synthesis activity.3 These findings are consistent with Lys329 as the site of enzyme intermediate formation and its involvement in catalysis as an active site residue.
The overall scheme for deoxyhypusine synthesis (Scheme 1, solid
arrows) postulates three imine-intermediates, formed in sequence. It is possible to write a plausible reaction scheme based on an imine
transfer mechanism, as has been extensively described by Jencks
(42-45) and demonstrated for enzymes utilizing pyridoxal phosphate as
a cofactor (43, 45, 46). When protonated, the imine group is highly
reactive and can undergo transimination (42-45). In the
case of deoxyhypusine synthase, the initial protonated imine could
arise through removal of a hydride ion (H) from
spermidine.4 Ensuing transimination
involving the initial imine (dehydrospermidine) and the enzyme results
in the formation of the next imine intermediate (Enz-Lys-N=CH(CH2)3NH2) with the
release of 1,3-diaminopropane. Subsequent transimination(s)
transfers the 4-carbon moiety from the unsaturated enzyme intermediate
to the eIF-5A precursor, forming an eIF-5A imine intermediate
(Pre-Lys-N=CH(CH2)3NH2). This
eIF-5A imine intermediate undergoes enzyme-catalyzed reduction to the deoxyhypusine form (eIF-5A(Dhp)) without exogenous reducing agent. The
origin of the hydride ion needed for this final step in the catalytic
reaction is not known. NADH generated during the dehydrogenation of
spermidine is its most likely source, either direct or indirect. On the
other hand, NADH does not reduce the unsaturated enzyme intermediate,
presumably due to an unfavorable orientation of the enzyme-imine with
respect to NADH. The absence of enzyme imine-intermediate reduction
precludes autoinactivation of the enzyme.
In the absence of eIF-5A precursor (Scheme 1, dashed
arrows), the unsaturated 4-carbon amine moiety would be retained
on the enzyme. This imine could slowly undergo cyclization to
1-pyrroline if its own free amino group acts as the
entering nucleophilic group to attack the imine at the secondary
nitrogen, as suggested previously (23). The time course of spermidine
cleavage in the absence of the protein substrate (Fig. 3) shows
indication of a delayed appearance of
1-pyrroline
relative to 1,3-diaminopropane. This implies that
1-pyrroline is not derived directly from the butylamine
of dehydrospermidine by nucleophilic attack on its own imine, but
rather from the enzyme-intermediate. It is possibly due to this
abortive cleavage that only a portion of the modified enzyme imine
intermediate could be trapped by reduction.
We had previously made a systematic study, using a series of spermidine analogs, to probe the active site of deoxyhypusine synthase (14, 15). From the observed efficiency of these inhibitors, we concluded that the orientation of the spermidine molecule, and specifically its secondary amine, N4, must be precisely defined in a cleft on the enzyme. The current studies, which indicate a specific relationship between lysine 329 of the human enzyme and N4-C5 of spermidine, are consistent with that suggestion. Determination of the x-ray crystal structure, currently under way5 should shed light on the spatial organization of spermidine and NAD at the active sites of the enzyme and permit tentative identification of amino acid residues involved in spermidine binding and catalysis.
We thank John Thompson, NIDR, for helpful comments on the manuscript.