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Yeast 2000; 16: 219±229.
Aminopeptidase yscCo-II: a new cobalt-dependent
aminopeptidase from yeastÐpuri®cation and
biochemical characterization
Area de BioquõÂmica, Centro de QuõÂmica del Instituto de Ciencias, Universidad AutoÂnoma de Puebla,
72000 Puebla, MeÂxico
Saccharomyces cerevisiae aminopeptidase yscCo-II (APCo-II) was puri®ed to apparent homogeneity by gel
®ltration, af®nity chromatography and anion-exchange chromatography. APCo-II is an hexameric cobaltdependent metallo-enzyme with an estimated native molecular mass of 290 kDa. Enzyme activity is only detected in
the presence of cobalt ions at pH 7.0. Substrate speci®city studies indicate that aminopeptidase yscCo-II cleaves
only basic N-terminal residues. PMSF, Cu2+, 1,10-phenanthroline and bestatin were found to be very strong
inhibitors of aminopeptidase yscCo-II activity. Kinetic studies indicated that the enzyme has a similar Km and KaCo
(activation constant of cobalt) and, following extraction of cobalt from the enzyme, activity was recovered only
after cobalt addition. Copyright # 2000 John Wiley & Sons, Ltd.
Ð Saccharomyces cerevisiae; protein degradation; cobalt-dependent aminopeptidase; protease
The unicellular eukaryote, Saccharomyces cerevisiae, is especially suited for revealing the biological role of peptidases, since it is easily accessible
to biochemical, genetic and molecular biological
techniques. It is gradually becoming clear that
peptide bond hydrolysis represents an essential
mechanism in the regulation of cell metabolism at
the post-translational level (SuaÂrez-Rendueles and
Wolf, 1988; Fuller et al., 1988; Heinemeyer et al.,
1991; Peters, 1994; Fujimura-Kamada et al., 1997;
Hubbard, 1998). Much work has already been
done on the two major proteolytic systems, the
lysosomal and the cytosolic, that are involved in
the degradation of proteins in S. cerevisiae
(Hirsch et al., 1989; Fujiwara et al., 1990; Lee
and Goldberg, 1996; Klionsky, 1998). The biochemical and genetic characteristics of the main
enzymes of each system, e.g. the vacuolar
exopeptidases and the multicatalytic enzyme
complex or proteasome, have been investigated
*Correspondence to: I. Herrera-Camacho, Area de BioquõÂmica
del Centro de QuõÂmica, Instituto de Ciencias, Universidad
AutoÂnoma de Puebla, Apartado Postal 1613, 72000 Puebla
Pue, MeÂxico. E-mail:
CCC 0749-503X/2000/030219±11$17.50
Copyright # 2000 John Wiley & Sons, Ltd.
very thoroughly (Achstetter and Wolf, 1985;
Rivett, 1993; Richter-Rouff and Wolf, 1993; Lee
and Goldberg, 1996; Groll et al., 1997; Gilon
et al., 1998). Transcriptional regulation of the
yeast vacuolar aminopeptidase yscI-encoding gene
(APE1) by carbon sources has been shown in very
recent years (Bordallo et al., 1995). Moreoever,
there is the interesting example of a eukaryotic
DNA-binding cysteine protease (Xu and Johnston, 1994), and the transcriptional regulation by
nutrient limitation of the CPS1 gene by means
of regulatory elements (Bordallo and SuarezRendueles, 1995).
A number of proteases in yeast have been
puri®ed and characterized, and their structural
genes analysed (for reviews, see Achstetter and
Wolf, 1985; Hirsch et al., 1989; Jones, 1991).
Recent studies have identi®ed methionine aminopeptidase (Chang et al., 1990) and aminopeptidase
yscXVI (Tisljar and Wolf, 1993); aminopeptidase
Y (Yasuhara et al., 1994). In the past few years, a
number of structural genes encoding yeast aminopeptidases have been cloned and sequenced, e.g.
the BLH1 gene, encoding thiol-aminopeptidase
(Enenkel and Wolf, 1993), and the aminopeptidase Y gene (Nishizawa et al., 1994).
Received 21 January 1998
Accepted 10 October 1999
Recently, cobalt-dependent enzymes have
become an increasingly studied research ®eld and
this has led to the description of new types of
proteolytic enzymes from both prokaryotes and
eukaryotes (Roderick and Matthews, 1993; Ar®n
et al., 1995). Indeed, there is evidence that most of
the aminopeptidase activities in yeast are stimulated, to a greater or lesser extent, by cobalt
(Co2+) (Hirsch et al., 1989; Herrera-Camacho,
1984; Chang et al., 1990; Tisljar and Wolf, 1993).
However, aminopeptidase yscCo is the only one
described to date as detectable only in the presence
of Co2+ (Achstetter et al., 1982). We will refer to
this enzyme as AP yscCo-I, following the nomenclature proposed by Achstetter and Wolf (1985).
In this paper, we report the puri®cation and
characterization of another aminopeptidase that is
only detectable in the presence of Co2+, which we
name aminopeptidase yscCo-II (APCo-II). Its
properties clearly differentiate this new aminopeptidase from all similar activities so far described in
Enzyme assays
All chromogenic peptide substrates were dissolved in water at a concentration of 10 mM, and
stored at x20uC. Aminopeptidase yscCo-II activity was determined routinely at 37uC with the
chromogenic substrate L-lysine-4-nitroanilide, and
the enzyme was pre-incubated in the presence of
Co2+ and a buffer for 10 min at 37uC, after the
start of the reaction with the substrate. An aliquot
(0.5 ml) of the test solution contained an enzyme
solution with 50 mM Tris±HCl buffer, pH=7.0,
0.5 mM CoCl2 and 1 mM of substrate solution. The
test was stopped by adding 0.5 ml 40 mM EDTA
(pH 8) and 20 mM chloroquine solution. The
release of 4-nitroaniline was measured in the
supernatant at 405 nm in a UV-160 Shimadzu
photometer. One milliunit (mU) of enzyme
activity is de®ned as the amount of enzyme that
releases 1 nmol product/min under test conditions.
The molar absorption coef®cient for 4-nitroaniline
at 405 nm is e405nm=9900 Mx1 cmx1; this value
was used for the calculation of enzyme activity.
Protein determination
The chromogenic peptide substrates, as well as
the proteinase inhibitors, were obtained from
Sigma (USA) or Bachem (Switzerland). All other
chemicals were of the highest purity available and
were purchased from Merck (Germany), Sigma
(USA) and Aldrich (USA). The yeast growth
medium was obtained from DIFCO (USA).
Matrices for gel chromatography were obtained
from Pharmacia Biotech (Sweden).
Protein was determined according to the
method of Sedmak and Grossberg (1977), using
crystalline bovine serum albumin as standard.
Puri®cation of aminopeptidase yscCo-II
All puri®cation steps were performed at 40uC.
Step 1. Crude extract supernatant
The preparation of crude extract supernatant
was performed using the conditions of SuaÂrezRendueles et al. (1981).
Yeast strain and growth conditions
Step 2. Filtration chromatography and molecular
Most experiments were performed with the
diploid Saccharomyces cerevisiae wild-type strain,
1022. S cerevisiae strain II-21 (MATa lap1 lap2
lap3 lap4 leu2±3,112), which lacks aminopeptidase
activity (Cuevas et al., 1989), was used as
reference; Dr Paz SuaÂrez-Rendueles kindly supplied this strain. Yeast cells were grown in a
minimal medium containing 0.7% Yeast Nitrogen
Base without amino acids, and 2% glucose, in a
rotary shaker at 32uC, and harvested towards the
end of the exponential growth phase (Abs600nm=
After dialysis of crude extract supernatant
against 20 mM MOPS/Tris, pH 7.0, 100 mM KCl,
5 mM N3Na (®ltration buffer), the extract was run
on a column (2.6r60 cm) of 320 ml Sephadex G200 (diluted with ®ltration buffer) at a linear ¯ow
rate of 10 ml/h. Fractions of 2.5 ml were collected
and those containing the highest activity pooled.
The following markers were used for molecular mass determinations of native APCo-II:
egg albumin (43 kDa), bovine serum albumin
(67 kDa), aldolase (158 kDa), catalase (232 kDa),
and ferritin (450 kDa).
Copyright # 2000 John Wiley & Sons, Ltd.
Yeast 2000; 16: 219±229.
Step 3. Af®nity chromatography
For this step, the af®nity adsorbant was
obtained by coupling the lys-phe dipeptide to
AH-Sepharose-4B, using the carbodiimide method
of Pharmacia-Biotech. Fractions (pooled according to step 2) were dialysed against 20 mM MOPSTris, pH 7.0 (af®nity buffer). They were then
applied to a Lys-phe-AH-Sepharose-4B (column
1.5r10 cm), equilibrated with af®nity buffer and
run with a buffer ¯ow rate of 12 ml/h. After
release, the protein was washed with 20 mM
acetate buffer, pH 5.5; APCo-II was eluted to
100 mM KCl in acetate buffer. Fractions of highest
activity were pooled and again dialysed against
af®nity buffer.
Step 4. Ion exchange chromatography
The APCo-II obtained in step 3 was applied to
the DEAE-Cellulose (column 2r15 cm) and run
in af®nity buffer with a ¯ow rate of 12 ml/h. After
release of the protein, the APCo-II was eluted to
50 mM KCl in af®nity buffer. Fractions of highest
activity were pooled, dialysed against af®nity
buffer, and stored at 40uC until used for the
characterization studies.
Polyacrylamide gel electrophoresis (PAGE)
After freeze-drying, a sample of puri®ed APCoII was loaded onto an SDS±PAGE gel (7%
running gel; 3.5% stacking gel). Electrophoresis
was performed under reducing conditions, as
described by Laemmli (1970), at 60 V (stacking
gel) and 100 V (running gel) in a Mini-Protean II
apparatus (Bio Rad). Protein staining with silver
was performed, as indicated by Morrisey (1981).
The Mr of APCo-II was determined from the
relative mobility of protein standards: phosphorylase b (97 kDa), bovine serum albumin (66 kDa),
egg albumin (45 kDa) and carbonic anhydrase
(29 kDa). Non-denaturing polyacrylamide gels
were prepared in the same manner as for
SDS±PAGE, but without the denaturing and
reducing conditions.
control was obtained with CoCl2 (1 mM) and
phen (1 mM) in af®nity buffer and after incubation
(18 h at 4uC), UV±VIS spectroscopy was performed.
The enzyme, after dialysis with phen, was
extensively dialysed against af®nity buffer with
frequent changes of buffer. For regeneration of
enzyme activity, the APCo-II solution (50 ml) was
pre-incubated in 100 ml of af®nity buffer containing 2.5 mM of the various metals for 20 min at
37uC. The enzyme assay was initiated by adding
the substrate, using the same conditions described
in the section on enzyme assays.
Kinetic studies
All kinetic studies were carried out at 37t0.1uC
using a D-160 Shimadzu-Spectrometer, employing
a Peltier system. Reaction rates were determined
in the continuous assay, and the release of 4nitroaniline was monitored by the change in
absorbance at 405 nm. The kinetic parameters
were determined using Lineweaver-Burk plots and
a program for regression and variance analysis.
Puri®cation and molecular properties
APCoII was puri®ed to homogeneity of S.
cerevisiae in three stages of chromatography.
Determination of APCo-II activity was made
after the ®rst stage of molecular-exclusion chromatography in Sephadex G-200, as shown in
Figure 1. The peak of activity of APCo-II was not
APCo-II metal(s) extraction and reactivation
Divalent ion(s) present in the puri®ed enzyme
were removed by dialysis for 18 h at 4uC of af®nity
buffer (20 ml) containing 5 mM 1,10-phenanthroline (phen). The dialysis buffer was concentrated
(20r) in a rotary evaporator and UV±VIS
spectral analysis was performed. An internal
Copyright # 2000 John Wiley & Sons, Ltd.
Figure 1. Gel permeation chromatography of aminopeptidase
yscCo-II on Sephadex G-200. Aminopeptidase activity without
cobalt ($) and with cobalt (+), protein concentration (#).
The crude extract supernatant was applied to the Sephadex G200 column. 100 ml aliquots of each fraction were employed for
activity assays. For details, see Materials and Methods.
Yeast 2000; 16: 219±229.
observed until CoCl2 was added to the lys-NA
substrate. When the elution position of APCo-II
on gel ®ltration was correlated with molecular
mass, a value of approximately 290 kDa was
obtained. The other peak to the right corresponds
primarily to AP-yscII, with an Mr of 85±100 kDa
(Frey and RoÈhm, 1978; Hirsch et al., 1989).
Given the recently described group of aminopeptidases, and as a check for the fact that this is a
different activity, the strain II-21 (MATa, lap1,
lap2, lap3, lap4, leu2±3, leu2±112), a mutant
in four aminopeptidase activities, was used as
reference. After chromatography in Sephadex G200, the Mr peak of 290 kDa of our APCo-II was
observed (data not shown), as well as a second
broad peak of less than 90 kDa, which most
probably corresponds to the recently described
aminopeptidases AP-yscXVI (Tisljar and Wolf,
1993) or AP-yscY (Yasuhara et al., 1994). The
AP-yscII activity was not detected in this strain
due to the lap2 mutation.
Subsequent steps in the puri®cation involved
subjecting the sample to a af®nity chromatography on a pre-equilibrated Lys-Phe-AH-Sepharose4B resin. Figure 2 shows that the APCo-II was
eluted at high ionic strength (0.1 M KCl) and
acidic pH (5.5). The enzyme binds very strongly to
the ligand and could not be separated by means of
the substrate lys-NA (data not shown). This
method is very selective, and approximately 95%
of the protein contaminants were eliminated
(Figure 2 and Table 1). Figure 3 shows the last
stage of puri®cation, ion exchange chromatogra-
Figure 2. Af®nity chromatography of aminopeptidase yscCoII on lysine-phenyl-AH-Sepharose 4B. Aminopeptidase activity
(2) and protein concentration (#). The aminopeptidase
fraction obtained by Sephadex G-200 chromatography was
applied to the lys-phe-AH-Sepharose 4B column. 100 ml
aliquots of each fraction were employed for activity assays.
For details, see Materials and Methods.
Copyright # 2000 John Wiley & Sons, Ltd.
phy, in which the APCo-II is bound to a cationic
resin and is eluted at low ion strength (0.05 M KCl)
whereas the rest of the protein contaminants
remain bound to the column. Table 1 shows the
stages of puri®cation, with a good yield of
27% and a puri®cation factor of 160. This factor
could be even higher if it were based on the
crude extract, which is impossible because of the
interference of the other aminopeptidases.
PAGE in native condition shows a single lane of
proteins (Figure 4, line 1) which, when cleaved and
incubated with the substrate and cobalt, reveals
the activity of APCo-II (data not shown). Running the sample under denaturing conditions
(PAGE±SDS), we observed a single band of
apparent molecular mass 48 kDa (Figure 4, line
3). Given the Mr of 290 kDa obtained by gel
®ltration (Sephadex G-200) and this single lane in
PAGE±SDS of 48 kDa, we ®nd that APCo-II is
a homo-hexamer protein. The PAGE and PAGE±
SDS experiments show that the enzyme has been
puri®ed to homogeneity.
APCo-II is rather unstable after storage of the
puri®ed enzyme at 4uC; 50% and 20% of the initial
activity was assayed after 4 and 8 days, respectively. The only way to keep it active for 2 months
at 4uC was to concentrate it and keep it
precipitated in a suspension with 4 M ammonium
sulphate in 20 mM MOPS/Tris, pH=7.
Properties of expression; dependence on cobalt
and pH
The activity of APCo-II was detected after the
®rst stage of size-exclusion chromatography. It is
impossible to detect the activity of APCo-II in a
crude soluble extract without interference because
of the other aminopeptidase activities that cleave
N-terminal lysine substrates, and because its
activity is stimulated by the presence of Co2+
(Achstetter et al., 1982; Herrera-Camacho, 1984;
Tisljar and Wolf, 1993; Yasuhara et al., 1994).
In order to determine how the expression of
APCo-II activity varies in terms of cellular
growth, soluble extracts were carried out in
different stages of growth, and after a molecularexclusion chromatography (Sephadex G-200),
enzymatic activity was determined. APCo-II
activity varies in the different stages of growth,
with a minimal expression (5%) in early exponential phase, increasing to maximum (100%) in the
late exponential phase, and decreasing (25%)
during the early stationary phase (data not
Yeast 2000; 16: 219±229.
Table 1.
Puri®cation of aminopeptidase yscCo-II.
Puri®cation step
Speci®c activity
Soluble extract
Sephadex G-200
shown). The optimum pH of APCo-II is 7
(Figure 5A), and its activity is strictly dependent
on Co2+, with a maximum of activity at
concentrations of 0.5 mM CoCl2 (Figure 5B). Due
to these characteristics, it is different from
the other known aminopeptidases; APCo-yscI
(Achstetter et al., 1982) expresses itself primarily
in the stationary phase, has a basic optimum pH
(8.5), and presents its maximum activity at
0.05±0.1 mM CoCl2. In the case of AP-yscII and
AP-yscY, enzymatic activities are not dependent
on Co2+; AP-yscII stimulates its activity with
Co2+ (Herrera-Camacho, 1984) and, in the case of
AP-Y, Co2+ has different effects (activating or
inhibiting), depending on the substrate used
(Yasuhara et al., 1994).
Substrate speci®city
breaking substrates with N-terminal basic amino
acids, such as lys-NA and arg-NA. With neutral
acidic terminal amino acids, as well as substrates
of carboxypeptidase yscY (BTNA) and substrates
of dipeptidyl-aminopeptidases (x-pro-NA), the
enzyme is unable to effect hydrolysis.
The Michaelis±Menten constant determined
with lys-NA and arg-NA as substrates and calculated from Lineweaver±Burk was found to be
0.16 mM for both. APCo-II hydrolyses lys-NA and
arg-NA at the same rate. In the case of APCo-I,
lys-NA is hydrolysed at a rate three times higher
than for arg-NA (Achstetter et al., 1982). In the
case of AP-Y, it hydrolyses a greater number
of substrates, with a preference for N-terminal
peptides and dipeptides, rather than N-terminal
amino acids (Yasuhara et al., 1994).
The effect of divalent metals on enzyme activity
The substrate speci®city of APCo-II, was
investigated using a broad spectrum of different
synthetic chromogenic aminoacyl derivatives.
Table 2 shows that APCo-II has a preference for
Given the fact that APCo-II activity can only be
detected in the presence of Co2+, and in order to
®nd out whether this activity were detectable when
Figure 3. Ion exchange chromatography of aminopeptidase
yscCo-II on DEAE±cellulose. Aminopeptidase activity (2)
and protein concentration (#). The aminopeptidase fraction
obtained by lys-phe-AH-Sepharose 4B chromatography was
applied to DEAE-cellulose column. 100 ml aliquots of each
fraction were employed for activity assays. For details, see
Materials and Methods.
Figure 4. Polyacrylamide gel electrophoresis (PAGE) of
puri®ed aminopeptidase yscCo-II. Line 1: APCo-II puri®ed
under native condition (PAGE). Line 2: molecular weight
markers in SDS-PAGE (97 kDa, phosphorylase b; 66 kDa,
bovine serum albumin; 45 kDa, egg albumin; 29 kDa, carbonic
anhydrase). Line 3: APCo-II puri®ed under denaturalized
condition (SDS±PAGE). The gel was stained with silver. For
details, see Materials and Methods.
Copyright # 2000 John Wiley & Sons, Ltd.
Yeast 2000; 16: 219±229.
Figure 5. Optimal conditions of aminopeptidase yscCo-II
activity. (A) Effect of pH on APCo-II activity. The following
buffers were used: Mes±Tris (5.5±6.5) (+), MOPS±Tris
(6.5±7.5) (&), Tris±HCl (7.5±9.0) ($). (B) Effect of Co2+on
the activity from APCo-II at pH=7.0. 0.5 mg protein,
corresponding to homogeneity of APCo-II, were employed as
the enzyme. For details of activity assays, see Materials and
the Co2+ is replaced by another divalent metal, a
study was carried out using different metal centres
in the presence and absence of Co2+ (Table 3).
Taking the activity shown with Co2+ as 100, we
found that no other divalent cation of those
assayed shows any important APCo-II activity
(Table 3, ®rst column). Ca2+ and Mg2+ recover a
minimal percentage (12%) of the activity, which
suggests that APCo-II is strictly dependent on
Co2+. On the other hand, when determining the
activity of APCo-II in the presence of Co2+, the
effect of the metal centres on enzyme activity can
be observed. Table 3, last column, shows that
the presence of Cu2+ totally inhibited APCo-II
activity. The divalent metal ions Cr2+, Zn2+ and
Ni2+ showed levels of inhibition of 83%, 70% and
Table 2. Substrate
Relative hydrolysis (%)
Activities against all substrates was tested with puri®ed
enzymes and are expressed relative to the rate of hydrolysis
of L-lysine-4-nitroanilide (100% was 4035 mU/mg). Activities
were measured at pH 7.0 with 1 mM substrate and 0.5 mM
CoCl2 at 37uC. Incubation for 30 min and enzyme activity was
determined as described in Materials and Methods.
Copyright # 2000 John Wiley & Sons, Ltd.
40%, respectively. Hg2+, Mg2+ and Mn2+ have
only a very weak effect as inhibitors. In the case
of Hg2+, the concentration must be increased ®ve
times (5 mM) in order to produce a total inhibition
of APCo-II activity. Different concentrations were
used for each metal (chlorides and sulphates), and
it could be observed that these have no signi®cant
effect on the APCo-II response to the different
It is interesting to observe how Zn2+, which is a
structural part of several enzymes, e.g. AP-yscI
(Metz and RoÈhm, 1976), AP-Y (Yasuhara, 1994)
and very probably also of AP-yscII (GarcõÂaAlvarez et al., 1991), behaves in others as an
inhibitor, e.g. APCo-I (Achstetter et al., 1982),
thiol-AP (Enenkel and Wolf, 1993), as well as this
enzyme, APCo-II. Cu2+ proves to be a strong
inhibitor of APCo-II as well as AP-II (HerreraCamacho, 1984) and AP-Y (Yasuhara et al.,
1994), whereas in the case of APCo-I, it has no
effect (Achstetter et al., 1982).
Table 3. Divalent metal ion dependency of aminopeptidase yscCo-II activity and their effect.
Cation added
(conc. 1 mM)
HgCl2 (5 mM)
Activity (%)
Without CoCl2
With 1 mM CoCl2
*Enzyme activity (control), 100% was 4019 mU/mg.
For the ion dependency of APCo-II, the enzyme puri®ed was
preincubated 10 min at 37uC in the presence of the respective
metal ion(s) and, for the effect of divalent cation, the enzyme
was pre-incubated with 1 mM CoCl2, after addition of
respective metal ion(s).
Yeast 2000; 16: 219±229.
Effect of protease inhibitors
The results of the study of APCo-II with
different inhibitors are shown in Table 4. On
assaying the effect of the chelating agents on
APCo-II activity, we observed that 1,10-phenanthroline showed an inhibition of 90% at a
concentration of 1 mM. At the same concentration,
the other chelating agents (ANT, EDTA and
chloroquine) show 50% inhibition. Inhibition with
1,10-phenanthroline is re-established when Co2+
is added (data not shown). Of the inhibitors of
microbial origin, bestatin at concentrations of
4 mg/ml has a strong inhibiting effect (close to
100%) on APCo-II. At the same concentration,
antipain and leupeptin produced a much weaker
inhibition of 15% and 25%, respectively. In order
to elicit an inhibition of 100% in the case of
antipain, a concentration of 2.5 times higher is
required. Bestatin proved to be a potent inhibitor
of APCo-II and, in contrast, does not have or
produce any effects on the activities of APCo-I,
Table 4.
thiol-AP, and AP-Y (Achstetter et al., 1982;
Enenkel and Wolf, 1993; Yasuhara et al., 1994).
Other inhibitors that interact directly with the
Zn2+ of metallo-aminopeptidases are hydroxamates (Holmes and Mattheus, 1981), in our study
leu-hydroxamate and lys-hydroxamate, which provoked an inhibition of 80% at 1 mM. With respect
to inhibitors of sulphhydryl groups, p-HMB at
concentrations of 1 mM did not have any effect on
APCo-II activity and requires higher concentrations in order to produce an inhibition of 90%.
This result is analogous to the effect produced
with metal Hg2+, which has great af®nity for the
sulphhydryl groups. PMSF and TLCK, inhibitors
of serine proteases, inhibit APCo-II at concentrations of 1 mM with 100% and 50%, respectively.
Given the strong inhibition with PMSF, it would
be plausible to believe that residues of serine,
tyrosine or threonine were involved in the enzyme
catalysis. PMSF, a potent inhibitor of APCo-II,
produces little or no effect on the activities of
APCo-I, thiol-AP and AP-Y (Achstetter et al.,
Effect of protease inhibitors on aminopeptidase yscCo-II activity.
Final concentration
Relative speci®c activity (%)
1 mM
5 mM
1 mM
5 mM
1 mM
5 mM
1 mM
5 mM
4 mg/ml
10 mg/ml
4 mg/ml
10 mg/ml
4 mg/ml
10 mg/ml
1 mM
5 mM
1 mM
5 mM
1 mM
5 mM
1 mM
5 mM
1 mM
5 mM
Nitrilotriacetic acid
¯uoride (PMSF)
Tosyl-lysine chloromethyl
ketone (TLCK)
Puri®ed aminopeptidase yscCo-II (4087 mU/mg) was incubated in the presence of the indicated inhibitors for 30 min and enzyme
activity was determined as described in Materials and Methods.
Copyright # 2000 John Wiley & Sons, Ltd.
Yeast 2000; 16: 219±229.
1982; Enenkel and Wolf, 1993; Yasuhara et al.,
APCo-II metal(s) extraction and regeneration of
enzyme activity
In order to examine the possible existence of a
metallic centre in APCo-II, the enzyme was
studied in presence of the chelating agent 1,10phenanthroline (phen). The endogenous metal
ion(s) was removed by dialysis against phen and
the resulting phen±Co coordination compound
was followed spectrophotometrically. The results
(shown in the Figure 6A) demonstrate that we
have detected a band at 510 nm, corresponding
to Co2+ d±d transitions (see control Co2+,
Figure 6B), and also a charge-transfer band
associated with UV±phen transitions (see control
phen, Figure 6D). The same pattern is obtained in
a phen±Co system which was used as an internal
control (Figure 6C). These results indicate that
APCo-II is a metallo-enzyme containing Co2+ in
its structure. On the other hand, the APCo-II
following interaction with phen (and after extensive dialysis against buffer) was inactive and
showed a 100% recovery of activity after Co2+
addition (Table 5). Other metal ions like Ca2+,
Cu2+, Mg2+, Mn2+, Ni2+ and Zn2+ could not
restore the enzyme activity. These results indicate
that the aminopeptidase activity is dependent only
on the divalent cation Co2+.
Kinetic studies of APCo-II
Since the enzyme under study is noticeably
cobalt-dependent, we studied its kinetic behaviour
by measuring the velocity of product formation as
a function of the substrate and of Co2+. The
APCo-II Lineweaver-Burk plot, in the presence of
different ®xed Co2+ concentrations (Figure 7),
shows a family of lines with differents slopes that
converge on the axis, 1/V0. The Vmax is independent of Co2+ concentrations; however, the Km
decreases as Co2+ increases. The maximum effect
of Co2+ on APCo-II activity was found at
0.5 mM, with no difference in Km differences
at higher concentrations. The measured Km and
Vmax values for APCo-II (at 0.5 mM of CoCl2) are
0.16 mM and 3.12 nmol/min, respectively. The
data, shown in the form of a Lineweaver±Burk
plot in Figure 7, are entirely consistent with a
`competitive activation' where the Co2+ is an
`essential activator' (Dixon and Webb, 1979). By
plotting [Co] vs. 1/Kmap, a Co-apparent activation
constant of Ka=0.28 mM was obtained (insert,
Figure 7), a value that is very similar to the Km
(0.16 mM), and supports the strict cobalt dependence of APCo-II.
Table 5.
Figure 6. Aminopeptidase yscCo-II metal(s) extraction. (A)
Spectrum of the concentrated dialisate after dialysis of APCoII with 1,10-phenanthroline. (B) CoCl2 spectrum. (C) (phen±
CoCl2) complex spectrum. (D) 1,10-phenanthroline spectrum.
For details, see Materials and Methods.
Copyright # 2000 John Wiley & Sons, Ltd.
Regeneration of enzyme activity by divalent
Cation added (0.5 mM)
Recovery activity (%)
Control (x phen)
Control (+ phen)
*100% corresponding to activity of the nontreated enzyme, and
is 4006 mU/mg.
The enzyme, after dialysis with 1,10-phenanthroline (phen),
was extensively dialysed against af®nity buffer. The APCo-II
solution (50 ml) was pre-incubated in the presence of 2.5 mM of
various divalent cations (100 ml) at 37uC for 20 min; the
substrate was then added and the enzyme activity determined
as described in Materials and Methods.
Yeast 2000; 16: 219±229.
1/Vo (nmol/min)
Km(Co 0.5mM) = 0.161 mM
Vmax = 3.12nmol/min
Figure 7. Kinetic study of APCo-II by Cobalt. The initial
data shown in Lineweaver±Burk transformation. CoCl2 concentration shown in the ®gure are (+) 0.1 mM, (+) 0.3 mM,
(&) 0.5 mM and (2) 1 mM. A secondary plot showing the
variation of (1/Kmap) with cobalt concentration is given in the
inset ®gure. For details of kinetic analyses, see Materials and
APCo-II activity is strictly dependent on Co2+,
which cannot replaced by any other divalent
cation tested. The APCo-II activity is speci®c for
N-terminal basic amino acid substrates. APCo-II
was puri®ed to homogeneity from the cell extract
by a three-step procedure. The puri®cation, as
estimated from the speci®c activity, was approximately 160-fold, with a yield of 27%. This value
was based on the original activity following
®ltration chromatography, since it is impossible
to obtain an accurate measure of the enzyme's
activity from the crude extract due to interference
from the other aminopeptidases.
The apparent molecular mass of the native
enzyme, calculated after gel ®ltration data, was
found to be 290 kDa. In contrast, under denaturing SDS±PAGE conditions, a single band of about
48 kDa was found. These data indicate that
aminopeptidase yscCo-II is a hexameric enzyme
made up of six identical subunits. Sequencing of
the amino-terminal region of the puri®ed enzyme,
using an Applied Biosystems 477A Protein
Sequencer, showed that the N-terminus of the
protein was blocked.
In the budding yeast S. cerevisiae, with the
exception of AP ysc-I (which has an Mr of
640 kDa; Frey and RoÈhm, 1978), APCo-II has
an Mr which, at 290 kDa, is considerably higher
than that of the other aminopeptidases described
in the literature (Hirsch et al., 1989; Chang et al.,
Copyright # 2000 John Wiley & Sons, Ltd.
1990; Tisljar and Wolf, 1993; Yasuhara et al.,
1994). The thiol aminopeptidase yeast (Enenkel
and Wolf, 1993) is the one that comes closest in
Mr (220 kDa); however, it differs clearly from
APCo-II, since the latter has a wider substrate
pro®le. Hydrolysing substrates with acidic, basic
and neutral N-terminal amino acids, APCo-II
is speci®c for basic N-terminal amino acids. Its
inhibitor pattern, on the other hand, is very
different, especially with bestatin and PMSF,
which are potent inhibitors of APCo-II, and
have no effect on thiol±AP (Enenkel and Wolf,
1993). The same inhibitors also have no signi®cant
effect on APCo-I (Achstetter et al., 1982), AP±
yscII (Herrera-Camacho, 1984) or on AP±Y
(Yasuhara et al., 1994). Given the strong inhibition with PMSF, it would be plausible to believe
that residues of serine, tyrosine or threonine were
involved in the enzyme catalysis.
Bestatin, an inhibitor of microbial origin, has
been described as an inhibitor of metallo-enzymes
(Salvesen and Nagase, 1989), interacting with the
metal centres of these enzymes. In studies with
aminopeptidases of Aeromonas, the metallic centre
that has previously been replaced by the enzyme's
Zn2+, and the direct interaction of bestatin
with Co2+, has been demonstrated (Wilkes and
Prescott, 1985). On the other hand, a study of
the absorption spectrum of cobalt(II)-substituted
Aeromonas aminopeptidase showed that it is
markedly perturbed by the presence of amino
acid-hydroxamates (Wilkes and Prescott, 1987).
Later studies of these metallo-enzyme inhibitors
claim that bestatin and the hydroxamates are
necessary to know the reaction mechanism of
APCo-II and its active centre. Due to inhibition
by chelating agents and reactivation by ion metals,
and the effect the other inhibitors, e.g. bestatin
and hydroxamate, APCo-II is classi®ed as a
metallo-enzyme. This seems to be the most
common class for yeast aminopeptidases.
A phen±Co coordination compound is formed
after incubation of APCo-II with 1,10-phenanthroline; this result is entirely consistent with the
presence of the cobalt in the molecular structure of
APCo-II. The APCo-II activity is strict cobaltdependent, having a Co-apparent activation constant very similar to the Km. The results of the
kinetic studies (Figure 7) clearly show a `competitive activation' in which Co2+ is an `essential
activation' (Dixon and Webb, 1979). The characteristic mechanism of this type of activation
established that the Co2+ binds to the free enzyme
Yeast 2000; 16: 219±229.
(E), and afterwards this complex (ECo) binds to
substrate (S), in a obligatory order of reaction,
written as:
E , ECo , ECoS
These results lend support to the cobalt±metalloenzyme character for APCo-II, and the involvement of the Co2+ metal in the catalytic function.
The recently described family of cobaltdependent enzymes, such as the methionine
aminopeptidases, is of particular interest. Ar®n
et al. (1995) described the structure of cobaltdependent methionine aminopeptidase from E.
coli and identi®ed the motif that binds the active
site cobalt ions (Roderick and Matthews, 1993).
APCo-II possibly belongs to this family, since
cobalt has been extracted from the structure of the
protein. However, to establish unambigously the
true coordination of cobalt in APCo-II and to
identify the amino acids of the active site, metal
analysis, X-ray crystallographic analysis, and
sequencing of the amino-terminal region of the
APCo-II is required. Such studies are under way
in our laboratory. The studies described here of the
new cobalt-dependent aminopeptidase, yscCo-II,
provide an important starting point for these
further investigations.
This work was supported in part by SEPDGICSA and CONACYT of MeÂxico. The
authors are especially grateful to Steve Oliver for
his help with English usage.
Achstetter T, Ehmann C, Wolf DH. 1982. Aminopeptidase Co, a new yeast peptidase. Biochem Biophys Res
Commun 109: 341±347.
Achstetter T, Wolf DH. 1985. Proteinases, proteolysis
and biological control in the yeast Saccharomyces
cerevisiae. Yeast 1: 139±157.
Ar®n SM, Kendall RL, Hall L, Weaver LH, Stewart
AE, Matthews BW, Bradshaw RA. 1995. Eukaryotic
methionyl aminopeptidases: two classes of cobaltdependent enzymes. Proc Natl Acad Sci U S A 92:
Bordallo J, SuaÂrez-Rendueles MP. 1995. Cis and transacting regulatory elements required for regulation of
the CPS1 gene in Saccharomyces cerevisiae. Mol Gen
Genet 246: 580±589.
Bordallo J, Cueva R, SuaÂrez-Rendueles MP. 1995.
Copyright # 2000 John Wiley & Sons, Ltd.
Transcriptional regulation of the yeast vacuolar
aminopeptidase yscI encoding gene (APE1) by
carbon sources. FEBS Lett 364: 13±16.
Chang YH, Teichert U, Smith JA. 1990. Puri®cation
and characterization of a methionine aminopeptidase
from Saccharomyces cerevisiae. J Biol Chem 265:
Cuevas R, GarcõÂa-Alvarez N, SuaÂrez-Rendueles MP.
1989. Yeast vacuolar aminopeptidase yscI. Isolation
and regulation of the APE1 (LAP4) structural gene.
FEBS Lett 259: 125±129.
Dixon M, Webb EC. 1979. Enzymes, 3rd edn. Academic
Press: New York.
Enenkel C, Wolf DH. 1993. BLH1 codes for a yeast
aminopeptidase, the equivalent of mammalian bleomycin hydrolase. J Biol Chem 268: 7036±7043.
Frey J, RoÈhm KH. 1978. Subcellular localization and
levels of aminopeptidases and dipeptidases in Saccharomyces cerevisiae. Biochim Biophys Acta 527:
Fujimura-Kamada K, Nouvet FJ, Michaelis S. 1997. A
novel membrane-associated metalloprotease, Ste24p,
is required for the ®rst step of NH2-terminal
processing of the yeast a-factor precursor. J Cell
Biol 136: 271±285.
Fujiwara T, Tanaka K, Orino E, Yoshimura T,
Kumatori A, Yamaguchi K, Shin K, Kakikazu A,
Nakanishi S, Ichihara A. 1990. Proteosomes are
essential for yeast proliferation. J Biol Chem 263:
Fuller RS, Sterne RE, Thorner J. 1988. Enzymes
required for yeast prohormone processing. Ann Rev
Physiol 50: 345±368.
GarcõÂa-Alvarez N, Cueva R, SuaÂrez-Rendueles MP.
1991. Molecular cloning of soluble aminopeptidases
from Saccharomyces cerevisiae. Sequence analysis
of aminopeptidase yscII, a putative zinc-metallopeptidase. Eur J Biochem 202: 993±1002.
Gilon T, Chomsky O, Kulka RG. 1998. Degradation
signals for ubiquitin system proteolysis in Saccharomyces cerevisiae. EMBO J 17: 2759±2766.
Groll M, Ditzel L, LoÈwe J, Stock D, Brochtler M,
Bartunik HD, Huber R. 1997. Structure of 20S
Ê resolution. Nature
proteasome from yeast at 2.4 A
386: 463±471.
Heinemeyer W, Kleinschmidt JA, Saidowsky J, Escher
C, Wolf DH. 1991. Proteinase yscE, the yeast
proteosome/multicatalytic proteinase: mutants unravel its necessity for cell survival. EMBO J 10:
Herrera-Camacho I. 1984. Mise en evidence de la
modulation directe des activities proteolytiques de la
levure Saccharomyces cerevisiae par des metabolites.
TheÁse de Docteur Biologie Cellulaire et Moleculaire,
Universite Pierre et Marie Curie, France.
Hirsch HH, SuaÂrez-Rendueles P, Wolf DH. 1989. Yeast
Saccharomyces cerevisiae proteinases: structure, characteristics and function. In Molecular and Cell
Yeast 2000; 16: 219±229.
Biology of Yeasts, Walton EF, Yarranton GT (eds).
Blackie: London; 134±200.
Holmes MA, Mattheus BW. 1981. Binding of hydroxamic acid inhibitors to crystalline thermolysin
suggest a pentacoordinate zinc intermediate in catalysis. Biochemistry 20: 6912±6921.
Hubbard SJ. 1998. The structural aspects of limited
proteolysis of native proteins. Biochem Biophys Acta
1382: 191±206.
Jones EE. 1991. Three proteolytic systems in the
yeast Saccharomyces cerevisiae. J Biol Chem 266:
Klionsky DJ. 1998. Non-classical protein sorting to the
yeast vacuole. J Biol Chem 273: 10807±10810.
Laemmli UK. 1970. Cleavage of structural proteins
during assembly of the head of bacteriophage T4.
Nature 227: 680±685.
Lee DH, Goldberg AL. 1996. Selective inhibitors of the
proteasome-dependent and vacuolar pathways of
protein degradation in Saccharomyces cerevisiae.
J Biol Chem 271: 27280±27284.
Metz G, RoÈhm KH. 1976. Yeast aminopeptidase I.
Chemical composition and catalytic properties. Biochem Biophys Acta 429: 933±949.
Morrisey JH. 1981. Silver stain for proteins in
polyacrylamide gels: a modi®ed procedure with
enhanced uniform sensitivity. Anal Biochem 17:
Nishizawa M, Yasuhara T, Nakai T, Fujiki Y, Ohashi
A. 1994. Molecular cloning of the aminopeptidase Y
gene of Saccharomyces cerevisiae. J Biol Chem 269:
Peters JM. 1994. Proteasomes: protein degradation
machines of the cell. Trends Biochem Soc 19: 377±382.
Richter-Rouff B, Wolf DH. 1993. Proteosome and cell
cycle. FEBS Lett 336: 34±36.
Copyright # 2000 John Wiley & Sons, Ltd.
Rivett AJ. 1993. Proteosomes: multicatalytic proteinase
complexes. Biochem J 291: 1±10.
Roderick SL, Matthews BW. 1993. Structure of the
cobalt-dependent methionine aminopeptidase from
Escherichia coli: a new type of proteolytic enzyme.
Biochemistry 32: 3907±3912.
Salvesen G, Nagase H. 1989. Inhibition of proteolytic
enzymes. In Proteolytic Enzymes, Beynon RJ, Bond
JS (eds). IRL Press: Oxford; 83±104.
Sedmak JJ, Grossberg SE. 1977. A rapid sensitive and
versatile for protein using Coomassie Brilliant Blue
G-250. Anal Biochem 79: 544±552.
SuaÂrez-Rendueles MP, Schwencke J, Garcia-Alvarez N,
Gascon S. 1981. A new X-prolyl-dipeptidyl aminopeptidase from yeast associated with a particulate
fraction. FEBS Lett 131: 296±300.
SuaÂrez-Rendueles MP, Wolf DH. 1988. Proteinase
function in yeast: biochemical and genetic approaches
to a central mechanism of post-translational
control in the eukaryotic cell. FEMS Microbiol Rev
54: 17±46.
Tisljar U, Wolf DH. 1993. Puri®cation and characterization of the cystinyl bond cleaving yeast aminopeptidase yscXVI. FEBS Lett 322: 191±196.
Wilkes HS, Prescott JM. 1985. The slow tight binding of
bestatin and amastatin to aminopeptidases. J Biol
Chem 260: 13154±13162.
Wilkes HS, Prescott JM. 1987. Hydroxamate-induced
spectral perturbations of cobalt Aeromonas aminopeptidase. J Biol Chem 262: 8621±8625.
Xu HE, Johnston SA. 1994. Yeast bleomycin hydrolase
is a DNA-binding cysteine protease. J Biol Chem 269:
Yasuhara T, Nakai T, Ohashi A. 1994. Aminopeptidase
Y, a new aminopeptidase from Saccharomyces
cerevisiae. J Biol Chem 269: 13644±13650.
Yeast 2000; 16: 219±229.
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