Department of Biochemistry and Molecular
Biology and Center for Metalloenzyme Studies, University of
Georgia, Athens, Georgia 30602,1 and
Department of Genetics, University of Utah, Salt Lake City,
Utah 841122
Proline dipeptidase (prolidase) was purified from cell extracts of
the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme is a homodimer (39.4 kDa per subunit) and as purified contains one cobalt atom per
subunit. Its catalytic activity also required the addition of
Co2+ ions (Kd, 0.24 mM), indicating
that the enzyme has a second metal ion binding site. Co2+
could be replaced by Mn2+ (resulting in a 25% decrease in
activity) but not by Mg2+, Ca2+,
Fe2+, Zn2+, Cu2+, or
Ni2+. The prolidase exhibited a narrow substrate
specificity and hydrolyzed only dipeptides with proline at the C
terminus and a nonpolar amino acid (Met, Leu, Val, Phe, or Ala) at the
N terminus. Optimal prolidase activity with Met-Pro as the substrate
occurred at a pH of 7.0 and a temperature of 100°C. The N-terminal
amino acid sequence of the purified prolidase was used to identify in
the P. furiosus genome database a putative
prolidase-encoding gene with a product corresponding to 349 amino
acids. This gene was expressed in Escherichia coli and the
recombinant protein was purified. Its properties, including molecular
mass, metal ion dependence, pH and temperature optima, substrate
specificity, and thermostability, were indistinguishable from those of
the native prolidase from P. furiosus. Furthermore, the
Km values for the substrate Met-Pro were
comparable for the native and recombinant forms, although the
recombinant enzyme exhibited a twofold greater Vmax value than the native protein. The amino
acid sequence of P. furiosus prolidase has significant
similarity with those of prolidases from mesophilic organisms, but the
enzyme differs from them in its substrate specificity, thermostability,
metal dependency, and response to inhibitors. The P. furiosus enzyme appears to be the second Co-containing member
(after methionine aminopeptidase) of the binuclear N-terminal
exopeptidase family.
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INTRODUCTION |
Pyrococcus furiosus is a
fermentative archaeon which grows optimally at temperatures near
100°C (26). Like many heterotrophic hyperthermophiles, it
utilizes proteins and peptides as growth substrates and produces
organic acids, CO2, and H2. Several enzymes involved in the catabolism of amino acids have been purified from P. furiosus (2), including aminotransferases
(3), glutamate dehydrogenase (34), 2-keto acid
oxidoreductases (31, 37, 38), and acetyl coenzyme A
synthetases (39). In addition, this organism produces
perhaps a dozen or more proteolytic-type enzymes, which are assumed to
generate small peptides from the protein-based growth substrates
(6, 8, 21, 23, 45). So far, three proteases have been
characterized from P. furiosus. These are a
membrane-associated serine protease (48), an intracellular protease with trypsin- and chymotrypsin-like activities (28, 29), and an intracellular endopeptidase that cleaves at prolyl residues (30). In addition, two proteases have been purified from other members of the family Thermococcales, including a
thiol protease from an unclassified species of Pyrococcus
(40) and a serine protease from Thermococcus
stetteri (33). To date, however, there have been no
reports on the properties of amino acid-yielding peptidases from
P. furiosus or related species. In order to further
understand the pathways of peptide metabolism in these organisms, we
examined P. furiosus for dipeptidase activities. Cell
extracts contained very high concentrations of prolidase, a
proline-specific dipeptidase, and the characterization of this enzyme
is reported herein.
Since prolyl residues confer a conformational constraint on a peptide
chain due to the cyclic nature of its pyrrolidine side group, only a
few proteases are known that are able to hydrolyze bonds adjacent to
proline (22, 49). These enzymes include (i) proline-specific
endopeptidase, which hydrolyzes peptides on the carboxyl side of prolyl
residues located internally within a polypeptide (-X--Pro-/-X-); (ii)
prolyl aminopeptidase, which cleaves the bond between any N-terminal
amino acid and a penultimate prolyl residue
(NH2-X-/-Pro--X-) in peptides of various lengths; (iii)
proline iminopeptidase, which catalyzes cleavage of unsubstituted N-terminal prolyl residues from dipeptides, tripeptides, and
polypeptides (Pro-/-X-); (iv) proline specific C-terminal exopeptidase
(-X--Pro-/-X-COOH), which releases an amino acid from the C terminus of
a peptide with a penultimate proline residue; and (v) prolidase, which
only cleaves dipeptides with proline at the C terminus
(NH2-X-/-Pro-COOH). These proline-specific enzymes are
thought to participate, in concert with other endo- and exopeptidases,
in the terminal degradation of intracellular proteins and may also
function in the recycling of proline.
Prolidase (iminodipeptidase, EC 3.4.3.7) is widespread in nature and
has been isolated from different mammalian tissues (12, 24,
44) as well as from bacteria such as species of Lactobacillus (10, 25) and Xanthomonas
(46). While the physiological role of prolidase in bacteria
is unclear, a deficiency of this enzyme in humans results in
abnormalities of the skin and other collagenous tissues
(43). Prolidase also has biotechnological applications. For
example, it has a potential use in the dairy industry as a
cheese-ripening agent (9) since proline release from
proline-containing peptides in cheese reduces bitterness. In addition,
it was recently reported (18) that prolidase and an enzyme
termed organophosphorus acid anhydrolase (OPAA, EC 3.1.8.1) appear to
be one and the same. OPAA hydrolyzes highly toxic, organophosphorus, acetylcholinesterase inhibitors, which include various chemical warfare
agents and pesticides. Such enzymes have been characterized from
various species of Pseudomonas, Flavobacterium,
and Alteromonas and also from eucaryotes (5, 17,
36). The sequence of the OPAA from Alteromonas sp.
strain JD6.5 shows similarity to that of human prolidase and, like the
latter enzyme, OPAA catalyzes the Mn2+-dependent hydrolysis
of X-Pro dipeptides but not of Y-Pro-X tripeptides (18). It
seems reasonable to conclude, therefore, that the natural function of OPAA involves peptide metabolism rather than
detoxification. Conversely, previously characterized prolidases may be
found to be biotechnologically relevant in detoxification strategies.
Prolidase has yet to be characterized from an archaeon or a
hyperthermophile. It was therefore of some interest to determine the
properties of this enzyme from P. furiosus. In addition, the gene encoding P. furiosus prolidase was cloned and expressed
in Escherichia coli, and this allowed a biochemical
comparison to be made between the native form (from P. furiosus) and the recombinant form.
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MATERIALS AND METHODS |
Growth of microorganisms.
P. furiosus (DSM 3638) was
grown at 95°C in a 500-liter fermentor with maltose as the carbon
source as described previously (13). E. coli
BL21(
DE3) (F
ompT [lon]
hsdS) was grown in a 100-liter fermentor at 37°C in Luria-Bertani medium supplemented with ampicillin (100 µg/ml) as
needed.
Enzyme assay.
The prolidase activity assay used was based on
the amount of proline liberated from the hydrolysis of dipeptides that
contain proline at the C terminus. The proline concentration was
determined by a modification of the colorimetric ninhydrin method of
Yaron and Mlynar (51). The ninhydrin reagent was prepared by
the addition of ninhydrin (3.0% [wt/vol]) to a mixture of 60%
(vol/vol) glacial acetic acid and 40% (vol/vol) phosphoric acid
followed by a 30-min incubation at 70°C (51). The assay
mixture (500 µl) for prolidase contained 50 mM MOPS
(3-[N-morpholino]propanesulfonic acid) buffer (pH 7.0), 4 mM Met-Pro (substrate), and 1.2 mM CoCl2 and was incubated at 100°C for 5 min. The reaction was initiated by addition of the
enzyme or extract. The mixture was incubated at 100°C for a further
10 min, and the reaction was stopped by the addition of glacial acetic
acid (500 µl) followed by the ninhydrin reagent (500 µl). After
heating at 100°C for 10 min, the solution was cooled to 23°C and
the absorption at 515 nm was determined with an extinction coefficient
of 4,570 M1 · cm1 for the
ninhydrin-proline complex. One unit of prolidase activity is defined as
the amount of enzyme that liberates one micromole of proline per minute
under these assay conditions.
Purification of P. furiosus prolidase.
Prolidase
was purified from P. furiosus under anaerobic conditions at
23°C. Frozen cells (500 g [wet weight]) were thawed in 1,800 ml of
50 mM Tris-HCl buffer (pH 8.0) containing lysozyme (1 mg/ml) and DNase
(10 µg/ml) and were lysed by incubation at 37°C for 2 h
followed by sonication (Branson 8200 sonicator) for 1 h. A cell
extract was obtained by ultracentrifugation at 50,000 × g for 2 h. The supernatant (1,800 ml) was loaded onto a
column (10 by 14 cm) of DEAE Fast Flow (Pharmacia, Piscataway, N.J.) equilibrated with 50 mM Tris (pH 8.0) containing 10% (vol/vol) glycerol. The column was eluted at a flow rate of 10 ml/min with a
10-liter linear gradient of 0 to 1.0 M NaCl in the same Tris-glycerol buffer. Prolidase activity was detected as 0.25 to 0.40 M NaCl was
applied to the column. The active fractions were combined (1,500 ml),
and solid ammonium sulfate was added to a final concentration of 1.5 M. This solution was applied to a column (3.5 by 10 cm) of phenyl
Sepharose (Pharmacia) equilibrated with Tris-glycerol buffer containing
1.5 M ammonium sulfate. The column was eluted with a gradient (1 liter)
from 1.5 to 0 M ammonium sulfate in the Tris-glycerol buffer at a flow
rate of 7 ml/min. Prolidase eluted as 0.45 to 0.78 M ammonium sulfate
was applied to the column. The prolidase-containing fractions (250 ml)
were concentrated to a volume of 7 ml by ultrafiltration (PM-30
membrane filter; Amicon, Beverly, Mass.) and applied to a column (3.5 by 60 cm) of Superdex-200 (Pharmacia) equilibrated with 50 mM Tris (pH
8.0) containing 0.5 M NaCl at a flow rate of 0.5 ml/min. The active fractions from the Superdex 200 column were applied to a column of
HiTrap-Q (1.6 by 2.5 cm; Pharmacia) equilibrated with 50 mM Tris (pH
8.0), and the enzyme was eluted with a gradient (100 ml) from 0 to 0.5 M NaCl in the same buffer at a flow rate of 4 ml/min. Fractions
containing prolidase activity (10 ml) eluted as 0.29 to 0.40 M NaCl was
applied and were stored at 
80°C until being required.
Cloning and expression of the prolidase-encoding gene.
Recombinant P. furiosus prolidase was obtained by PCR
amplification of the P. furiosus prolidase gene and
subsequent cloning of this gene into the T7-polymerase-driven
expression vector pET-21b (Novagen, Milwaukee, Wis.). For the PCR
amplification of the prolidase gene, two primers were designed. Primer
1 (ATAGGATCCGGTGAGGAGGTTGTATGAAAGAAAGACTTGAA; Stratagene, La
Jolla, Calif.) contained an engineered BamHI site and spans
from 21 to +6 on the coding strand. Primer 2 (ATAGGATCCGGTGAGGAGGTTGTATGAAAGAAAGAC; Stratagene) had an
engineered NotI site and corresponds to sequence ranging
from +1511 to +1541 on the noncoding strand. PCR amplification was
performed with native P. furiosus DNA polymerase and a
Robocycler 40 (Stratagene) programmed for 39 cycles, each cycle
consisting of denaturation at 95°C for 5 min, annealing at 52°C for
2 min, and extension at 72°C for 5 min. The resultant 1.5-kb
prolidase gene was first subcloned into the blunt end SrfI
site in the vector pCR-Script (Stratagene) to yield plasmid pProl. The
prolidase insert DNA contained in plasmid pProl was then sequenced to
ensure that no mutations were present in the gene. The prolidase gene was then excised from plasmid pProl by restriction digest with the
enzymes BamHI and NotI (Stratagene) and cloned
into the BamHI and NotI sites in expression
vector pET-21b, resulting in plasmid pET-Prol.
For expression of recombinant prolidase, plasmid pET-Prol was
transformed into E. coli BL21(DE3), which has
isopropyl-
-D-thiogalactopyranoside (IPTG)-inducible expression of T7-RNA polymerase. Prolidase
was produced in a culture of BL21(DE3)-pET-Prol grown in a 100-liter fermentor at 37°C. Expression of the plasmid borne prolidase gene was
induced with the addition of IPTG (1 mM) when the culture reached an
optical density of 1.0. The induced culture was incubated for 4 h
prior to the harvesting of the cells.
Purification of recombinant prolidase.
Recombinant prolidase
was purified in three steps. IPTG-induced BL21(DE3)-pET-Prol cells
(10 g [wet weight]) were suspended in 10 ml of 50 mM Tris-HCl, pH
8.0, containing benzamidine HCl (0.5 mg/ml). The cell suspension was
passed through a French pressure cell (20,000 lb/in2)
twice. The lysed extract was centrifuged at 39,000 × g
for 1 h to remove any cellular debris, and the supernatant was
diluted to 300 ml with 50 mM Tris-HCl, pH 8.0. Solid ammonium sulfate was slowly added with stirring to a final concentration of 1.5 M, and
the solution was applied to a column (3.5 cm by 10 cm) of phenyl
Sepharose (Pharmacia) equilibrated with the same buffer at a flow rate
of 7 ml/min. The bound protein was eluted with a gradient (1,000 ml)
from 1.5 to 0 M ammonium sulfate in 50 mM Tris-HCl, and the recombinant
prolidase was eluted as 0.67 to 1.0 M ammonium sulfate was applied. The
prolidase-containing fractions were incubated at 100°C for 2.5 h, and denatured E. coli proteins were removed by
centrifugation at 27,000 × g for 30 min. The
supernatant was diluted threefold with 50 mM Tris-HCl, pH 8.0, as it
was applied to a column of HiTrap-Q (1.6 by 2.5 cm; Pharmacia)
equilibrated with 50 mM Tris-HCl, pH 8.0. A gradient (100 ml) from 0 to
0.5 M NaCl in the same buffer was applied to the column. The prolidase eluted between 0.25 and 0.37 M NaCl and was stored at 80°C until being required.
Other methods.
Molecular weights were estimated by gel
filtration with a column (1 by 27 cm) of Superdex 200 (Pharmacia LKB)
with amylase (200,000), alcohol dehydrogenase (150,000), and bovine
serum albumin (66,000) as standard proteins. Sodium dodecyl sulfate
(SDS)-gel electrophoresis was performed using 12% polyacrylamide by
the method of Laemmli (35). Protein concentrations were
determined by the Bradford method (11) with bovine serum
albumin as the standard. To determine metal content, exogenous metal
ions were removed from the prolidase by either dialyzing (membrane
cutoff, 8 kDa) the sample against 100 volumes of 50 mM
N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid (EPPS), pH 8.0, containing 0.5 M NaCl or by gel filtration with a
Superdex 200 column. A complete metal analysis (31 elements) was
obtained by plasma emission spectroscopy with a Jarrel Ash Plasma Comp
750 instrument at the Chemical Analysis Laboratory of the University of
Georgia. The NH2-terminal sequences of the native and
recombinant prolidases were determined by using an Applied Biosystems
Model 477 sequencer in the Molecular Genetics Instrumentation Facility
(MGIF) of the University of Georgia. Samples were electroblotted onto
polyvinylidene difluoride protein-sequencing membranes (Stratagene)
from SDS-electrophoresis gels by using a Bio-Rad electroblotting
system. Electroblotting was carried out in 10 mM
3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) buffer, pH 11.0, containing methanol (10% [vol/vol]) for 1 h at 50 V. Both
strands of the P. furiosus prolidase gene present in plasmid
pET-Prol were sequenced in their entirety by the MGIF of the University
of Georgia. DNA sequence was analyzed using the computer software
programs Genetics Computer Group (University of Wisconsin, Madison) and
MacVector (International Biotechnologies, Inc., New Haven, Conn.).
Nucleotide sequence accession number.
The DNA sequence of
the prolidase gene is available from GenBank under accession no.
AF060010.
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RESULTS |
Purification of P. furiosus prolidase.
Extracts of
P. furiosus cells grown with maltose as the primary carbon
source contained high prolidase activity (approximately 2.3 U/mg at
100°C) with the dipeptide Met-Pro as the substrate. For comparison,
this specific activity is more than 100-fold higher than the prolidase
activity found in cell extracts of Lactobacillus casei grown
on casein (25) and 200-fold higher than that in cell
extracts of Xanthomonas maltophilia grown in nutrient broth (46). The prolidase of P. furiosus appeared not
to be regulated, as the specific activities of extracts of cells grown
with yeast extract (5.0 g/liter) and maltose (1.0 g/liter), with yeast
extract (5.0 g/liter), peptone (5.0 g/liter) and maltose (1.0 g/liter), or with yeast extract (5.0 g/liter) and maltose (5.0 g/liter) were
similar (2.5 ± 0.3 U/mg). Since maltose-grown cells are routinely used in our laboratory to purify various O2-sensitive,
oxidoreductase-type enzymes (see reference 2 for an
example) these cells were also used for prolidase purification. In
addition, the procedure was carried out under anaerobic conditions, not
because the prolidase was sensitive to O2, but to allow the
purification of enzymes that are from the same batch of P. furiosus cells.
Prolidase activity was not detected in the culture supernatant during
either log- or stationary-phase growth of P. furiosus or in
the membrane fraction of a cell extract. The activity was present only
in the soluble fraction, indicating that the enzyme is a cytoplasmic
protein. The results of a typical purification are summarized in Table
1. The enzyme was purified 274-fold with a yield of 4% and a specific activity of approximately 630 U/mg. It
therefore constitutes approximately 0.36% of total cytoplasmic protein. When the purified prolidase was treated with SDS sample buffer
at 80°C for 10 min prior to electrophoresis, it migrated as a single
band corresponding to a molecular mass of 51 kDa. However, when treated
at 100°C for 30 min, the protein band migrated with a molecular mass
of 42 kDa (see Fig. 1). Presumably, the former conditions result in a partially denatured protein which is
retarded in the electrophoretic gel. The prolidase which was eluted
from a gel filtration column corresponded to a molecular mass of
100 ± 10 kDa. This result, together with the electrophoretic data, suggests that the enzyme is a homodimer.
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FIG. 1.
SDS-12% polyacrylamide gel electrophoresis of purified
N- and R-prol. Lane 1, molecular mass markers (kilodaltons): myosin
(200), -galactosidase (116), phosphorylase b (97), bovine
serum albumin (66.3), glutamic dehydrogenase (55.4), lactate
dehydrogenase (36.5), carbonic anhydrase (31); lane 2, R-prol; lane 3, N-prol.
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The N-terminal amino acid sequence of the native prolidase was
MKERLEKLVKFMDEN. This sequence was used to search the genomic sequence
database of P. furiosus, which is nearing completion by the
use of multiplex sequencing methods (19). A gene was located
whose translated N-terminal region matched exactly the sequence
obtained from the enzyme. It consisted of 1,047 bp and encoded a
protein of 349 residues with a calculated molecular mass of 39.4 kDa
(Fig. 2). The latter value is slightly
lower than that (42 kDa) obtained from the SDS-gel analysis, suggesting that the protein is not completely denatured under the conditions used.
The enzyme also appears to exhibit nonideal behavior when subjected to
gel filtration, since the molecular mass estimated by that method (100 kDa) is higher than that expected (78.8 kDa) for a homodimeric protein.
A mass of 39.4 kDa for the prolidase subunit was used in all
calculations.
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FIG. 2.
The 1,047-bp gene encoding P. furiosus
prolidase and the deduced amino acid sequence (348 amino acids) are
shown. A putative TATA box is indicated in boldface. The ribosomal
binding site is underlined, and the translation start site is marked by
an arrow.
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Purification of recombinant P. furiosus prolidase.
The production of prolidase protein was successfully induced in
E. coli cells in the presence of IPTG with maximal induction (as determined by prolidase activity) after a 4-h period of induction at 37°C (data not shown). P. furiosus prolidase activity
could be identified in cell extracts of E. coli both by
high-temperature (100°C) enzyme assays, which eliminated the host
cell prolidase activity, as well as by the appearance of a protein band
corresponding to the size of prolidase (42 kDa) after SDS-gel analysis
of cell extracts (data not shown). The specific activity of the
prolidase in the recombinant E. coli cells was approximately
90 U/mg, which is about 40-fold higher than that present in cell
extracts of P. furiosus (under the same assay conditions
when Met-Pro was used as a substrate at 100°C). The results of a
typical purification of the recombinant prolidase from a cell extract
of E. coli BL21(DE3)-pET-Prol are summarized in Table
2. The enzyme was purified in three steps with a specific activity increase of about 15-fold and a recovery of
54%. It constituted approximately 6.7% of the total cellular protein.
However, the specific activity of the purified recombinant form (~ 1,300 U/mg) was about twofold greater than that of the native enzyme
from P. furiosus. The reasons for this are unclear at
present (see below). Nevertheless, the recombinant prolidase was
indistinguishable from the native protein from P. furiosus when analyzed by SDS-gel electrophoresis, and N-terminal amino acid
sequence analysis showed that it contained the same first 15 residues
(MKERLEKLVKFMDEN). It should also be noted that the nucleotide
sequence of the gene encoding the prolidase was identical to that
obtained from the genomic database.
Physical properties of native and recombinant prolidases.
Both
the native prolidase purified from P. furiosus (hereafter
referred to as N-prol) and the recombinant prolidase obtained from
E. coli (hereafter referred to as R-prol) were analyzed for metals. The only ones present in significant amounts (>0.1
g-atom/39.4-kDa subunit) were cobalt and zinc. Both R-prol and N-prol
contained 1.0 ± 0.3 (mean ± standard deviation) g-atoms of
Co/mol of subunit (data from four and eight different prolidase
samples, respectively). These data indicate that the difference in the
specific activities of N- and R-prol under standard assay conditions
was not due to a difference in Co content. R-prol was also purified as
described above but with all buffers containing 1 mM CoCl2.
The resulting enzyme, after gel filtration (Superdex 200) to remove
exogenous Co, also contained 1.0 (± 0.2) g-atoms of Co/subunit.
However, this and all other prolidase preparations tested were inactive unless Co2+ ions were added to the assay medium, suggesting
that both the recombinant and native forms of the enzyme require
occupancy of a second (or more) Co2+ site per subunit for
activity and that the two sites have very different affinities (see
below). Chemical analysis of R-prol and N-prol also revealed the
presence of significant but variable amounts of zinc (1.5 ± 1.0 and 2.0 ± 0.5 g-atoms/subunit from 4 and 12 determinations,
respectively). However, there was no correlation between the zinc
content and the specific activity of an enzyme preparation (whether R-
or N-prol), indicating that the zinc is nonspecifically bound. This was
confirmed by treating R-prol (containing 2.0 ± 0.1 g-atoms of
Zn/subunit) with EDTA (5 mM in 50 mM EPPS buffer, pH 8.0) for 1 h
at 23°C, followed by gel filtration. The resulting enzyme contained
only 0.3 ± 0.1 g-atoms of Zn/subunit, but its specific activity
under standard assay conditions was not affected by the chelation
treatment.
N-prol was very thermostable, with no loss in activity when a sample
(0.3 mg/ml in 100 mM MOPS, pH 7.0) was incubated in a sealed vial for
12 h at 100°C. However, the stability was dependent upon protein
concentration, as the same enzyme preparation at a concentration of
0.003 mg/ml lost 50% of its activity after a 4-h incubation at that
temperature. R-prol was apparently less thermostable than the native
form but also exhibited a concentration-dependent response. The time
required for a 50% loss in the activity (t50%) of R-prol at 100°C at a concentration of 0.3 or 0.003 mg/ml was 3 or
1 h, respectively. With R-prol at a concentration of 1.5 mg/ml,
the optimal pH for stability was 7.0, with t50% values at 100°C decreasing from 7.8 to 0.7, 1.3, 6.4 and 4.5 h at pH 2.0 (100 mM glycine-HCl), 4.5 (100 mM sodium acetate), 8.0 (100 mM EPPS), and 10.0 (100 mM CAPS), respectively. Hence, the enzyme was
much more stable under alkaline conditions than it was in acidic media.
Addition of Co2+ ions (1 mM) to either enzyme form during
the various heat treatments did not affect the results. These data also
demonstrate that both the recombinant and native forms of the prolidase
are stable under the routine assay conditions (10 min at 100°C).
Catalytic properties of native and recombinant prolidases.
The
catalytic activities of N- and R-prol showed virtually identical
responses to changes in temperature and pH (Fig.
3). Both showed a pH optimum at 7.0 and a
temperature optimum of 
100°C. Indeed, under the assay conditions
used, neither form exhibited detectable activity at temperatures of
40°C. From the temperature-dependent data, the calculated
activation energies for N- and R-prol are 11.9 ± 67.34 and
10.3 ± 77.86 kcal/mol, respectively. All assay reaction mixtures
used for both N- and R-prol included 1.2 mM Co2+, and these
ions could not be replaced with other divalent (Mg2+,
Ca2+, Fe2+, Zn2+, Cu2+,
or Ni2+) or monovalent (Na+ or K+)
cations (no activity was detected), with the exception of
Mn2+. The effects of Co2+ and Mn2+
concentrations on the activities of N- and R-prol are shown in Fig.
4. The two enzyme forms showed very
similar responses, with maximal activities at concentrations of 1.2 mM
CoCl2 and 1.6 mM MnCl2, with the latter
supporting approximately 75% of the activity of the former. However,
both cations caused some inhibition when added above their optimal
concentration (Fig. 4). The apparent association constants for
Co2+ and Mn2+ were 0.24 and 0.62 mM for N-prol
and 0.5 and 0.66 mM for R-prol, respectively. When N-prol was incubated
with 1 mM Co2+ ions and then assayed in the absence of the
metal, there was no difference in the specific activity (compared to
standard conditions where Co2+ ions are included in the
assay medium). However, when the sample was preincubated with 1 mM
Co2+ and then dialyzed (against 3,000 volumes of 50 mM MOPS
buffer, pH 7.0), only 5% of the activity remained. Addition of
Co2+ ions (1.0 mM) to the dialyzed enzyme completely
restored enzyme activity (assayed in the absence of Co2+
ions), but when EDTA (1 mM) was also added, no activity was detected. It therefore appears that the purified forms of both N- and R-prol contain one tightly bound Co2+ ion per subunit, but that
one (or more) additional cation(s), which can be either
Co2+ or Mn2+, is required for activity.
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FIG. 3.
The effects of pH (A) and temperature (B) on the
activities of N-prol (squares) and R-prol (circles). The assay mixtures
contained prolidase (0.015 µg), Met-Pro (4 mM), and CoCl2
(1.2 mM). For determination of the effects of pH, the following buffers
(each at 50 mM) were used: sodium acetate, pH 5.0; bis-Tris-HCl, pH
6.0; MOPS, pH 7.0; EPPS, pH 8.0; CHES
(2-[N-cyclohexylamino]-ethanesulfonic
acid), pH 9.0; and CAPS, pH 10.0. The assays were carried out at
100°C. For determination of the effects of temperature, the buffer
used was 50 mM MOPS, pH 7.0. An N-prol activity level of 100%
corresponds to 600 U/mg while 100% R-prol activity corresponds to
1,250 U/mg (with Met-Pro as substrate and measured at 100°C).
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FIG. 4.
The effects of Co2+ and Mn2+
ions on the activities of N- and R-prol. The assay mixtures contained
0.02 µg of N-prol (solid symbols) or R-prol (open symbols), Met-Pro
(4 mM), and various concentrations of either CoCl2
(squares) or MnCl2 (circles).
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Prolidase was identified by its ability to hydrolyze the dipeptide
Met-Pro, and this substrate was used in all routine assays. The
activities of N- and R-prol with other peptides are shown in Table
3. Both gave virtually identical results.
They only hydrolyzed dipeptides with Pro at the C terminus (not the N
terminus), and the nature of the N-terminal residue was critical, with
significant activity occurring only with peptides containing nonpolar
amino acids. Kinetic analyses were conducted using N-Prol with the five prolyl-containing dipeptides with which it showed significant activity
(Table 3). All exhibited normal Michaelis-Menton-type kinetics, and the
kinetic constants, shown in Table 4, were
calculated from linear double-reciprocal plots. The affinities of the
enzyme for Met-Pro and Leu-Pro, the two substrates with which it showed the highest kcat/Km
values, are lower than might have been anticipated, but the
kcat/Km values for all
five dipeptides are not too dissimilar, suggesting perhaps that all are
of physiological significance. The affinity of R-prol for Met-Pro is
comparable to that of the native enzyme (Table 4) although the
kcat value is more than twofold higher,
consistent with the results obtained under standard assay conditions.
The activities of the native and recombinant forms were not
significantly affected when they were treated (at 25°C for 30 min,
prior to assaying under standard conditions) with any of the following
protease (thiol or serine) inhibitors (each at 1 mM final
concentration): iodoacetate, ES-64
(L-transepoxysuccinyl-leucylamido (4-guanido) butane),
N-ethyl maleimide, phenyl methane sulfonyl fluoride, or
diisopropylphosphofluoride.
| |
DISCUSSION |
P. furiosus contains significant intracellular
concentrations of prolidase, an enzyme that appears to hydrolyze only
dipeptides that contain Pro at the C terminus and a nonpolar residue at
the N terminus. This finding is consistent with the proteolytic nature of the organism, although the rather high Km
values (3 to 20 mM) determined for the enzyme's substrates suggest
that such dipeptides must be present at significant intracellular
concentrations in vivo. The gene encoding the enzyme was successfully
expressed in E. coli although, surprisingly, the recombinant
form had a higher specific activity than the native prolidase. Why this
is the case is unclear since the molecular weight (as judged by SDS-gel electrophoresis), N-terminal amino acid sequence, activation by metal
ions (Co2+ and Mn2+), temperature and pH
dependence, and substrate specificities of the two enzyme forms were
indistinguishable. The fact that the recombinant form was slightly less
thermostable than the native protein suggests that it may not be
completely folded, and perhaps this additional flexibility leads to
enhanced catalytic activity.
Kinetic and metal analyses indicated that P. furiosus
prolidase (both native and recombinant) has at least two binding sites per subunit for Co2+ ions. One appears to be an integral
part of the protein (and not removed by purification or dialysis) while
the other(s) has an association constant of ~0.3 mM and is essential
for catalysis. In this regard the P. furiosus enzyme
resembles certain members of the broad class of binuclear
metallohydrolases represented by the N-terminal exopeptidases, the
active sites of which also contain two metal ions that typically differ
in their exchange kinetics (50). Prototypical members of
this family are bovine leucine aminopeptidase (15) and
Aeromonas proteolytica aminopeptidase (20), each
of which contains two Zn2+ ions per catalytic unit. The
zinc atoms of the A. proteolyticus enzyme can be replaced in
vitro with cobalt, and apparently like P. furiosus
prolidase, this amino peptidase can be prepared containing just one
metal ion per active site (7). The only naturally occurring,
cobalt-dependent members of this enzyme class are the methionine
aminopeptidases (4), and these contain two Co2+
ions per active site. The crystal structure of the E. coli
enzyme (42) shows that the two cobalt ions are coordinated
by five amino acid residues (Asp97, Asp108, His171, Glu204, and
Glu235), and all five are conserved in the sequences of the other
methionine aminopeptidases (16, 41, 47). Interestingly,
although the amino acid sequence of P. furiosus prolidase
shows no significant similarity with those of methionine
aminopeptidases, all five of the cobalt-coordinating residues are
conserved in the P. furiosus enzyme (Asp209, Asp220, His280,
Glu313, and Glu327) (Fig. 5). Clearly,
spectroscopic and structural analyses of the P. furiosus enzyme will be required to determine if it does, in fact, contain a
binuclear cobalt site and if the site is analogous to that of the
methionine aminopeptidase. Such studies are in progress. In this
regard, the ability of P. furiosus prolidase to be activated by Mn2+ ions suggests that an active enzyme form containing
a Co-Mn binuclear center should be possible, and this should facilitate
the interpretation of spectroscopic data. On the other hand, both R-
and N-prol also contained significant amounts of zinc. This appears to
be nonspecifically bound, however, as Zn2+ ions did not
support enzyme activity (in place of Co2+ or
Mn2+ ions) and typical zinc-binding motifs, e.g., HEXXH
(32), were not present in the sequence.
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|
FIG. 5.
Alignment of the amino acid sequence of P. furiosus prolidase with other prolidases (Prol),
Alteromonas OPAA, and E. coli methionine
aminopeptidase (MAP). The GenBank accession numbers for the prolidases
other than the one sequenced in this work are as follows: P46545,
L. delbrueckii prolidase; P15034, E. coli
prolidase; U56398, Alteromonas OPAA; and P07906, E. coli methionine aminopeptidase. Identical residues are designated
by the gray shading while similar residues are boxed. The five residues
that are ligands to the binuclear cobalt site in the subunit of
E. coli methionine aminopeptidase are indicated by
asterisks.
|
|
Database searches indicated that the amino acid sequence of P. furiosus prolidase showed significant similarity to the sequences of all known prolidases and to a putative prolidase in the genome sequence of the archaeon Methanococcus jannaschii
(14). The P. furiosus enzyme showed overall
similarities of 69, 61, 58, 56, and 53% with respect to the prolidases
from M. jannaschii, Lactobacillus delbrueckii,
Haemophilus influenzae, and E. coli and the human
prolidase, respectively. In all of these enzymes, there are three
extended regions of identity in the C terminus, YFXHXLGHXVGLEVHE
(P. furiosus prolidase residues 277 to 292), GMVXTIEPGIY
(residues 307 to 317), and GGVRIED (residues 322 to 328). These regions
contain three of the five putative Co2+-binding residues
mentioned above for the P. furiosus enzyme and presumably
form the active-site residues in all of these enzymes. Thus, of the
five residues in P. furiosus enzyme that are proposed to
bind Co2+ ions, all of them are conserved in the other
prolidases (Fig. 5).
The prolidase from P. furiosus represents the first such
enzyme to be purified from either an archaeon or a hyperthermophile. All other prolidases are from mesophilic sources (10, 12, 24, 25,
27, 46, 52) and are maximally active at temperatures up to
55°C. As might be expected, the P. furiosus enzyme is by far the most thermostable example of a prolidase, with a temperature optimum above 100°C and no loss of activity after 12 h at this temperature. Indeed, it is one of the most thermostable enzymes known
of any type (1). Like the P. furiosus enzyme, the
prolidases from X. maltophilia (46) and from
eucaryotic sources (guinea pig brain [12], human
erythrocytes [24], and bovine intestine [52]) are dimers (although their subunits are larger,
50 to 58 kDa), whereas the enzymes from Lactobacillus lactis
(10) and L. casei (25) are monomers
(~42 kDa). So far, however, P. furiosus prolidase is the
only one that has been shown to contain cobalt as an integral part of
the protein. The enzymes from L. casei, X. maltophilia, and human beings are activated by Mn2+
although their metal contents have not been reported, while it is not
known if the prolidases from Aureobacterium esteraromaticum (27), L. lactis, guinea pig brain and bovine
intestine contain a metal center or if they are activated by any metal
ion. However, the high similarities in the sequences of these enzymes
(Fig. 5), and the conservation of the five putative Co-binding residues found in P. furiosus, suggest that they all contain a
similar and presumably binuclear metal center, even though the nature of the metal (Mn2+ or Co2+) may not be the
same.
On the other hand, there are differences in substrate specificities of
the various prolidases. For example, the P. furiosus enzyme
is the only one which utilizes only dipeptides with proline at the C
(but not the N) terminus. The prolidases of L. lactis and
A. esteraromaticum hydrolyze dipeptides with Pro at either the N- or C-terminal position, and the enzymes from L. casei
and guinea pig brain efficiently cleave some dipeptides with no prolyl residue. Similarly, the mesophilic prolidases (with the exception of
the L. casei enzyme) are inhibited by cysteine protease
inhibitors such as N-ethyl maleimide and
p-chloromercuribenzoate, suggesting that a reactive cysteine
is required for catalysis. However, the P. furiosus enzyme
was not inhibited by these reagents. Only one Cys residue is present in
this prolidase, and this residue is not conserved in any of the
mesophilic prolidases, which contain between 2 (M. jannaschii) and 12 (human) Cys residues per subunit. Thus, it
would seem unlikely that a Cys residue is directly involved in the
catalytic mechanism of any of these prolidases.
The only other enzyme with which the P. furiosus prolidase
showed significant sequence similarity was that of OPAA from
Alteromonas (51% similarity and 24% identity). This enzyme
is capable of hydrolyzing a variety of toxic organophosphorus compounds
(17). Although OPAA is a monomeric enzyme, in contrast to
dimeric P. furiosus prolidase, its activity is dependent
upon Mn2+ ions. Moreover, the five residues proposed to
coordinate the binuclear metal center in the P. furiosus
enzyme are also conserved in the sequence of OPAA (Fig. 4). Thus, while
OPAA has a broad substrate specificity and is capable of hydrolyzing
P-F, P-C and P-O bonds (17), it exhibits comparable activity
with prolidase-type dipeptides such as Leu-Pro and Ala-Pro.
Furthermore, like the P. furiosus enzyme, it does not
hydrolyze tri- or tetrapeptides or dipeptides with Pro at the N
terminus (18). Clearly, these two enzymes are closely
related, and the effectiveness of the P. furiosus enzyme in
degrading organophosphorus compounds is currently being explored.
This research was supported by grants from the Office of
Industrial Technology of the Department of Energy
(SW994-19/RXE-7-17039) and the National Science Foundation
(BCS-9632657).
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Abstract
Introduction
