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INTRODUCTION |
The rising interest, both basic and applied, in
aminopeptidases (EC 3.4.11) from different sources has led to
the discovery of a number of enzymes that differ from each other in
cellular location, catalytic mechanism, and substrate specificity
(1-3). The majority of bacterial monoaminopeptidases are intracellular or membrane-bound metalloenzymes (1). Based on substrate specificity, bacterial monoaminopeptidases can be divided into two basic categories, specific aminopeptidases, which release only a limited number of amino
acids, and those that are able to liberate a relatively broad spectrum
of N-terminal amino acid residues (1).
Proline and glycine are among the most difficult residues for
aminopeptidases to hydrolyze because of their unique structures. Proline is unusual because of its cyclic structure, and glycine is
identified by the lack of a side chain. Nature has developed a family
of enzymes that recognize proline exclusively (4). A monoaminopeptidase
that preferentially releases glycine with high efficiency has not yet
been described and thus would be of high interest.
In this report we describe a novel extracellular monoaminopeptidase
from Sphingomonas capsulata. This enzyme has a clear
preference for N-terminal glycine and alanine. Because of this
characteristic, this monoaminopeptidase has the potential to
significantly enhance the degree of protein hydrolysis (5) when used as
a supplement to endoproteases and other exopeptidases.
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EXPERIMENTAL PROCEDURES |
Materials
Chemicals used as buffers and reagents were commercial products
of at least reagent grade. para-Nitroanilides of
L-amino acids and peptide substrates were from Sigma or
Bachem. Pentapeptide amides were synthesized at the Core Laboratories
(Louisiana State University). S. capsulata strain IFO
12533 was purchased from the Institute for Fermentation (Osaka, Japan).
A Whatman glass microfiber 2.7-µm filter and Nalgene Filterware
equipped with a 0.45-µm filter were used for filtering buffers and
supernatants. Protein purification was performed on an Amersham
Pharmacia Biotech fast performance liquid chromatography device
with column supports and resins from the same. Ultrafiltration units
(10-, 180-, and 350-ml) and membranes were from Amicon. The
Tricine1 gels and
polyvinylidene difluoride membranes used in the peptide separation and
sequencing process were from Novex. The molecular weight of proteins
was estimated using Novex Multi-Mark pre-stained and Mark 12 SDS-PAGE
markers. Endoproteinase Glu-C (V8 protease) was obtained from Roche
Molecular Biochemicals. Assays were performed on a THERMOmax microplate
reader, Shimadzu spectrophotometer UV160U, or Hewlett Packard Series
1050 HPLC system with column supports from Vydac, Inc. The protein
sequencer used was from Applied Biosystems (model 476A). The sequencing
reagents were purchased from PerkinElmer Life Sciences.
Purification of 66-kDa Aminopeptidase
Cultivation of S. capsulata strain IFO 12533 was
performed for 15 h at 31 °C, 250 rpm, and initial pH value of
7.45 in 1.5 liters of medium composed per liter of 10 g of
bactopeptone, 5 g of yeast extract, 3 g of NaCl, 2 g of
K2HPO4, 0.1 g of
MgSO4·7H2O, and 5 g of glucose
(autoclaved separately).
The culture broth supernatant (~1 liter) was obtained by initial
centrifugation followed by filtration using a Whatman glass microfiber
and Nalgene Filterware 0.22-µm filters consecutively. The filtrate
was concentrated using an Amicon spiral ultrafiltration system equipped
with a PM-10 ultrafiltration membrane. The sample was equilibrated with
10 mM phosphate buffer, pH 6.0, until the conductivity and
pH value were equal to the loading buffer, 50 mM MES, pH
6.0. The filtered solution was loaded onto a 24 × 390-mm column
containing ~180 ml of SP-Sepharose fast flow,
pre-equilibrated with 50 mM MES buffer, pH 6.0. Protein
with aminopeptidase activity was eluted with a 240-ml gradient from 0 to 0.2 M NaCl in 50 mM MES buffer, pH 6.0. Fractions with enzymatic activity toward Ala-pNA were pooled, desalted
using a PM-10 membrane, and equilibrated with 20 mM
phosphate buffer, pH 7.0.
The pooled solution was then loaded onto a 20-ml Amersham Pharmacia
Biotech Mono Q Bead column pre-equilibrated with 20 mM phosphate buffer, pH 7.0. Protein with aminopeptidase activity did not
bind to the column and was collected in the flow-through. The
flow-through was concentrated using a PM-10 membrane system as above,
and the pH value was adjusted to 6.0 with 70 mM acetate buffer, pH 4.0.
The concentrated flow-through was loaded onto a 1.0-ml Amersham
Pharmacia Biotech Mono S column that had been pre-equilibrated with 50 mM MES buffer, pH 6.0. The aminopeptidase was eluted with a
60-ml gradient from 0 to 0.2 M NaCl in 50 mM
MES buffer, pH 6.0. The fractions with significant activity were then
pooled, concentrated, and equilibrated with 50 mM phosphate
buffer, pH 7.0, containing 0.5 M
(NH4)2SO4 using a PM-10 membrane as above.
Finally, the concentrated sample was loaded onto an Amersham Pharmacia
Biotech phenyl Superose 5/5 pre-packed 7 × 50-mm column pre-equilibrated with 50 mM phosphate buffer, pH 7.0, containing 0.5 M
(NH4)2SO4. Protein with
aminopeptidase activity was then eluted with a 30-ml gradient from 0.5 to 0 M (NH4)2SO4 in 50 mM phosphate buffer, pH 7.0. Fractions containing
aminopeptidase activity were analyzed by SDS-PAGE and then pooled.
Periplasmic Extraction of S. capsulata
A culture broth was grown in a 500-ml shake flask under the
conditions described above. A sample was centrifuged, and the whole
cells were separated from the supernatant. The harvested cells were
then resuspended in 40 ml of 10 mM phosphate buffer, pH
6.0, which contained 1 mg/ml of lysozyme and 0.1% Triton X-100, and
stirred for 2 h. The resulting sample was centrifuged, and the
supernatant was analyzed.
N-terminal and Internal Amino Acid Sequences of Wild Type
Aminopeptidase
SDS-PAGE electrophoresis and transblotting were performed
according to the Novex instruction manual. Stained protein bands were
excised and sequenced following the instruction manual from PerkinElmer
Life Sciences. Data were collected and analyzed on a Macintosh IIsi
using Applied Biosystems 610 Data Analysis Software.
To generate peptide fragments of the enzyme to obtain internal
sequences, the protein was cleaved with cyanogen bromide by reconstituting a dried sample of the purified protein in 70% formic acid with a few crystals of cyanogen bromide and incubating for 18 h at room temperature in the dark. The peptide fragments were separated
by SDS-PAGE electrophoresis using a 10-20% Novex Tricine gel and
sequenced as described above. To generate additional peptide fragments,
the purified aminopeptidase was digested using endoproteinase Glu-C (V8
protease). A 200-µg sample of purified aminopeptidase was
equilibrated in 0.125 M Tris-HCl, pH 6.7, to a final volume of 60 µl, and 7.5 µl of 0.125 M Tris-HCl, pH 6.7, containing 2.5% w/v of SDS was added. The sample was boiled for 2 min
and then allowed to cool to room temperature. Then 10 µl of a 400 µg/ml solution of endoproteinase Glu-C was added, and the resulting sample was incubated at 37 °C for 2 h. To this solution, 45 µl of Novex 2× Tricine sample buffer was added. The peptide
fragments were separated by SDS-PAGE electrophoresis using a 10-20%
Novex Tricine gel and sequenced as described above.
Metal Content Analysis
The metal content was determined by atomic absorption
spectroscopy using a PerkinElmer Life Sciences 2380 atomic absorption spectrophotometer equipped with an HGA-400 graphite furnace. Each measurement was performed in triplicate. The concentration was determined by running a standard curve of known metal concentration.
Physicochemical Characterization
Assay--
Aminopeptidase activity was monitored using Ala-pNA
as the substrate. A stock solution of 100 mg/ml Ala-pNA in dimethyl
sulfoxide was diluted with 50 mM phosphate buffer, pH 7.5, to a concentration of 2 mg/ml. The reaction of the aminopeptidase with
the para-nitroanilide was initiated when a 10-50-µl
aliquot of the enzyme solution was added to 200 µl of the substrate
solution in a microtiter plate well. Initial rates of hydrolysis of the
para-nitroanilide were monitored at 405 nm at room
temperature using a THERMOmax microplate reader.
Substrate Specificity and Inhibition Study--
Stock solutions
of para-nitroanilides of various amino acids in dimethyl
sulfoxide (100 mg/ml) were diluted with 50 mM MOPS buffer,
pH 7.5, to concentrations of 2 mg/ml. Where the substrates were
incompletely soluble, their suspensions were used. The reaction of the
S. capsulata aminopeptidase with each
para-nitroanilide was initiated when an aliquot (10-µl) of
the enzyme solution in 50 mM phosphate, pH 7.0, was added
to 190 µl of a substrate solution in a 96-well microtiter plate.
Hydrolysis of the para-nitroanilides was monitored at 405 nm
and 25 °C.
Enzymatic hydrolysis of pentapeptides (see structures in Table II) was
performed at pH 7.5 in 50 mM MOPS buffer at 21 °C. Concentrations of the peptides in the incubation mixtures were between
1.10 and 1.14 mM. To stop the reactions, 50-µl aliquots of the incubation mixture were mixed with 50 µl of 0.1 M
HCl. The resulting samples were analyzed for free amino acids by
reverse-phase HPLC (6). The concentrations of the peptide solutions
were determined using an extinction coefficient of 1440 M
1 cm
1 at 280 nm for the
tyrosine residue.
The aminopeptidase was incubated for 2.5 h in 50 mM
MOPS buffer, pH 7.5, that contained no inhibitor, 1 mM
EDTA, 1 mM o-phenanthroline, or 1 mM
phenylmethylsulfonyl fluoride. Following the incubation, the enzyme
samples were assayed using Ala-pNA as described above.
pH Optimum--
An aliquot (20-µl) of a stock solution of
Ala-pNA in dimethyl sulfoxide (100 mg/ml) was diluted with 980-µl
aliquots of sodium acetate-Tris-HCl buffer (0.125 M) that
had different pH values between 5.0 and 8.5. The resulting pH value of
the substrate solutions was measured. A stock solution (0.05 mg/ml)of
the aminopeptidase in 50 mM phosphate buffer was diluted
5-fold by 10 mM Tris-HCl buffer, pH 7.5. The reaction
mixture contained 200 µl of the substrate solution and 10 µl of an
enzyme solution at room temperature.
Temperature Optimum--
An aliquot (970-µl) of 50 mM MOPS buffer, pH 7.5, was incubated for 15 min at the
chosen temperature, which was maintained using a thermojacket (Shimadzu
cell positioner CPS-240A) of the spectrophotometer. Then, 30 µl of
100 mg/ml Ala-pNA in Me2SO was added. The reaction was
initiated by adding 7 µl of enzyme solution. Initial velocities were
monitored over a 2-min period at 405 nm.
Thermal Stability--
An enzyme aliquot (10-µl) was added to
190 µl of 50 mM phosphate buffer, pH 7.5, which had been
preincubated for 30 min at the chosen temperature. The sample was
placed on ice after a 20-min incubation. The samples were then assayed
at room temperature following the protocol shown above.
Sequential Release of N-terminal Amino Acid Residues from a
Natural Peptide--
Leucine enkephalin was dissolved in 1 ml of 50 mM MOPS buffer, pH 7.5, to a final concentration of 1 mg/ml. Enzymatic hydrolysis was initiated by 8.3 µg of S. capsulata aminopeptidase. After incubation at room temperature
(21 °C), aliquots of the incubation mixture were added to 0.1 N HCl to terminate the reaction. The free amino acids of
the sample were analyzed by reverse-phase HPLC (6).
Cloning
Construction of a Genomic DNA Library--
Genomic DNA was
isolated from S. capsulata IFO 12533 using a Qiagen Tip-500
column as per the manufacturer's instructions (7). The library was
constructed by ligating Sau 3A partially digested
(5-7-kilobase) S. capsulata IFO 12533 chromosomal DNA into
the BamHI sites of the vector pSJ1678 (8) (Fig.
1) and transformed into Escherichia
coli XL1 Blue MR supercompetent cells (Stratagene,
Inc.).
Polymerase Chain Reaction Amplification of Aminopeptidase Coding
Sequences--
The following primers were synthesized based on amino
acid sequence data obtained from peptide fragments obtained following cyanogen bromide and V8 protease digestion of the purified
aminopeptidase. Forward primer, 5'-GCRTCRTANGCRTCNCC-3'; reverse
primer, 5'-ACYTTYTCRTCYTTRTC-3' (R = A or G, Y= C or T, N = A
or G or C or T).
Amplification reactions were prepared in a 50-µl volume with 50 pmol
of forward and reverse primers, 1 µg of S. capsulata IFO
12533 chromosomal DNA as template, 1× polymerase chain reaction buffer
(PerkinElmer Life Sciences), 200 µM each of dATP, dCTP, dGTP, and dTTP, and 0.5 units of AmpliTaq Gold (PerkinElmer Life Sciences). Reactions were incubated in a Stratagene Robocycler 40 (Stratagene) programmed for 1 cycle at 95 °C for 10 min, 35 cycles
each at 95 °C for 1 min, 44 °C for 1 min, and 72 °C for 1 min,
and 1 cycle at 72 °C for 7 min. The resulting product of ~190 base
pairs was cloned into vector pCR2.1/TOPO as per the manufacturer's
instructions (Invitrogen, Inc.).
Identification of Aminopeptidase Clones--
The genomic
S. capsulata IFO 12533 library was screened by colony
hybridization using a polymerase chain reaction-generated probe with
the Genius chemiluminescent system (Roche Molecular Biochemicals) as
per the manufacturer's instructions.
DNA Sequence Analysis of S. capsulata IFO 12533 Aminopeptidase
Gene--
DNA sequencing of two aminopeptidase-containing clones,
pMRT004.1-7 and pMRT004.1-14, was performed with an Applied
Biosystems model 373A automated DNA sequencer (Applied Biosystems,
Inc.) on both strands using (a) the Primer Island
Transposition method (Applied Biosystems, Inc.) as per the
manufacturer's instructions and (b) the primer walking
technique using dye-terminator chemistry (9). Oligonucleotide
sequencing primers were synthesized by Operon Technologies, Inc.
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RESULTS |
Localization and Purification of the Native Enzyme--
The
aminopeptidase was obtained from the supernatant of S. capsulata IFO 12533. Periplasmic extraction of the whole cells was
also performed, but the enzyme was not present in the extract. It is
evident from this result that the S. capsulata
aminopeptidase is a secreted enzyme. The purification led to a
protein that migrated as a single band of 66 kDa on SDS-PAGE (Fig.
2)

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Fig. 2.
SDS-PAGE of purified S. capsulata
aminopeptidase stained with Coomassie Brilliant Blue R-250.
Left lane, molecular mass marker (from top to
bottom) 200, 116.3, 97.4, 66.3, 55.4, 36.5, 31, 21.5, and
14.4 kDa, respectively; right lane, purified S. capsulata aminopeptidase.
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Physicochemical Properties of the Enzyme--
A specific activity
of 105 units/mg was determined for Ala-pNA under the condition
described above, assuming that the A280 of a 1 mg/ml solution of the aminopeptidase is 1.89. The theoretical extinction coefficient of the enzyme was calculated based on the deduced protein sequence (10).
We were able to determine the kinetic parameters for Ala-pNA
(kcat = 7600 ± 850 min
1, and
Km = 14 ± 2 mM) and alanine
-naphthylamide (kcat = 860 ± 90 min
1, and Km = 6.7 ± 1.1 mM). However, the kinetic parameters for the other amino
acid para-nitroanilides and
-naphthylamides could not be
accurately measured. This can be attributed to a combination of large
Km values, as well as poor solubility of the
synthetic substrates. In terms of relative activity, the S. capsulata aminopeptidase preferably hydrolyzes alanine
para-nitroanilide. It also demonstrates high efficacy on
para-nitroanilides of leucine, methionine, glycine,
and aspartic and glutamic acids (Table
I).
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Table I
Relative activity of the aminopeptidase with para-nitroanilides of
different amino acids
MOPS buffer, 0.125 mM, pH 7.5. [E]0 = 0.0194 µM. Concentration of the substrates is 7.7 mM.
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The lower estimation of turnover numbers of the S. capsulata
aminopeptidase on a series of pentapeptide amides with different N-terminal amino acids are shown in Table
II. The enzyme exhibited the highest
"turnover" on the pentapeptide amide with N-terminal glycine,
followed by alanine, leucine, glutamate, and proline.
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Table II
Substrate specificity of recombinant S. capsulata aminopeptidase
expressed in Bacillus subtilis toward XAPYK-amide pentapeptides
For conditions, see "Experimental Procedures".
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A study of the hydrolysis of several natural peptides, catalyzed by the
S. capsulata aminopeptidase, revealed that the enzyme is
capable of hydrolyzing a variety of peptide bonds. Among the bonds most
readily hydrolyzed was a Gly-Gly bond (Table
III). This is very unusual. The Gly-Gly
bond is extremely resistant to enzymatic hydrolysis, probably because
of a lack of side chain groups on both the N-terminal and penultimate
amino acids. The S. capsulata aminopeptidase also hydrolyzed
the peptides YAGFL and EALELARGAIFQA-amide with obvious
"bottlenecks" at phenylalanine (data not shown). It is important to
stress that it also releases N-terminal proline (Table II). This
aminopeptidase, meanwhile, does not split off N-terminal amino acids
with a penultimate proline and thus possesses no proline aminopeptidase
activity.
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Table III
Time release of N-terminal amino acids from leucine enkephalin (2.50 nmol) by recombinant S. capsulata aminopeptidase expressed in B. subtilis
For the specific conditions, see "Physicochemical
Characterization."
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Only o-phenanthroline demonstrated an inhibitory effect
among the class-specific inhibitors tested. In this case, the residual activity was found to be 4% of the initial. Neither EDTA nor
phenylmethylsulfonyl fluoride influenced the performance of the enzyme.
Certain inorganic anions that form salts with zinc of low solubility
were found to have inhibitory properties. Among the compounds examined
were phosphate, ferrocyanide, and iodate. The Ki values for these ions were determined to be 3.0, 4.2, and 11 mM, respectively. There is a direct correlation between the
Ki value and the solubility product of the
particular anion with zinc.
A plot of the relative activity of S. capsulata
aminopeptidase in the hydrolysis of Ala-pNA gave a typical bell-shaped
pH dependence with a sharp optimum at pH 7.2-7.4. An incubation of the
enzyme for 20 min at 45 and 50 °C led to losses of 40 and 100%
activity, respectively. The optimal temperature for the activity was
determined to be 43 °C.
Sequencing of the Wild Type S. capsulata Aminopeptidase--
The
N-terminal sequence of the 66-kDa homogeneous protein was determined to
be blocked. Digestion of the protein with cyanogen bromide resulted in
fragments with molecular masses of 42, 30, 17, 15, 10, 6, and 4 kDa. The N-terminal sequence of the 10-kDa fragment was determined to
be AVNGDAYDADKLKGAITNAKTGNPGAGRPI. The N-terminal sequences of the
other bands were inconclusive. The digestion with endoproteinase Glu-C
resulted in the generation of peptides with molecular masses of 40, 30, 25, 22, 20, 17, 10, 6, 5, and 4 kDa. The sequence
FKDEPNPYDKARMADAKVLSLFNSLGVTLDKDGKV was obtained from the 22- and
17-kDa peptide fragments. The other bands were either not sequenced, or
the results were inconclusive. The obtained protein sequences did not
demonstrate homology to any known aminopeptidases.
Sequence Analysis of the DNA Encoding S. capsulata
Aminopeptidase--
Screening of the S. capsulata IFO 12533 genomic library produced five colonies that exhibited strong
hybridization signals with the probe. Two plasmids carrying the
aminopeptidase gene were sequenced, and one was confirmed to contain
the entire aminopeptidase gene. Sequence analysis of the plasmid
containing the aminopeptidase gene revealed an open reading frame of
2010 nucleotides, encoding a protein of 670 amino acids. The G+C
content of this open reading frame was 65%. Based on the rules of von
Heijne (11), the first 32 amino acids likely comprise a secretory
signal peptide that directs the nascent polypeptide into the periplasm.
The calculated molecular mass of the primary translation product
determined from the deduced amino acid sequence of the S. capsulata aminopeptidase was 70.6 kDa, which is consistent with the estimation of 66 kDa based on the mobility of the purified protein
on SDS-PAGE. The zinc binding motif HEXXH, which is present in a number of metallopeptidase families (12), was identified in the
primary sequence (Fig. 3). A BLAST search
of the S. capsulata aminopeptidase against known data bases
(EBI, Swiss-Prot, GenBankTM, and GENESEQ) revealed no
significant homology to any known aminopeptidases. The highest percent
identity (23%) was observed with a hypothetical 67-kDa protein from
Synechocystis sp.
The presence of one atom of zinc per enzyme molecule was detected by
atomic absorption spectroscopy. This method also indicated four iron
atoms per enzyme molecule in a homogeneous preparation of the S. capsulata aminopeptidase. There were no detectable levels of
cobalt or calcium.
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DISCUSSION |
Bacteria hydrolyze different proteins to acquire essential amino
acids from the pool of free amino acids and peptides. Certain organisms, such as the nutritionally fastidious Lactococcus
lactis, apply a non-direct mechanism and employ a cascade of endo-
and exopeptidases to release N-terminal glycine (1, 13). Others, such
as Xanthomonas citri, have developed a more rational method, producing aminopeptidases with broad substrate specificity (14). In
this report, we show that S. capsulata secretes a unique
enzyme that preferably liberates N-terminal glycine. Because of a
combination of large Km values for both
para-nitroanilides and
-naphthylamides coinciding with
low solubilities for these compounds, it is impossible to carry out a
comprehensive study of the substrate specificity of the S. capsulata aminopeptidase utilizing kinetic data for these
artificial substrates. Nonetheless, a few remarks can be made. This
enzyme apparently discriminates similar amino acids effectively
releasing leucine but not isoleucine or valine (Table I); results for
alanine para-nitroanilide and
-naphthylamide unequivocally show that the structure of the leaving group for a
substrate affects both kcat and
Km. Taking this into account and expecting that the
substrate preference of an aminopeptidase toward derivatives of amino
acids, such as para-nitroanilides, and natural peptides can
be substantially different (15), the study of the hydrolysis of
non-protected peptides catalyzed by the S. capsulata
aminopeptidase was warranted. Comparative analysis of the Pro-pNA and
Pro-Ala-Pro-Tyr-Lys-amide highlights the misleading role of the
para-nitroanilide group. Apparently, it hinders the binding
of at least some amino acid residues, for example proline, by the
enzyme. More importantly, the S. capsulata aminopeptidase demonstrates an unusual substrate pattern with the order of preference Gly > Ala > Leu in terms of relative activity (Table II). A
plausible explanation for these features could be a catalytic pocket
that is not deep and exhibits very limited flexibility.
Leucine aminopeptidases are widely distributed in bacteria (1).
Normally they are completely passive toward glycine (16). There are a
few bacterial aminopeptidases described in the literature that
demonstrate a reasonable ability to release alanine. The alanine-specific aminopeptidase N from E. coli (17)
was not shown to release N-terminal glycine. The bimolecular constant for the thiol aminopeptidase from X. citri was almost
40-fold greater for alanine
-naphthylamide in comparison to glycine
-naphthylamide (14). It is highly unlikely that this enzyme is
capable of cleaving a Gly-Gly bond.
Aminopeptidase from S. capsulata occupies a unique niche
among bacterial proteases. This is the first reported enzyme that hydrolyzes natural peptides with bimolecular constant values that are
similar for glycine and alanine or probably even higher for glycine.
This extraordinary substrate preference is undoubtedly exhibited in the
hydrolysis of leucine enkephalin catalyzed by the S. capsulata aminopeptidase (Table III). A quick and complete release
of tyrosine and both glycine residues from this peptide was observed
after a 1-h reaction, yet the Phe-Leu bond was nearly untouched. This
clearly demonstrates the substantially higher catalytic efficacy of
this enzyme for amino acids with small, rather than large, side chains.
A high kcat value, at least 5400 min
1, for releasing glycine is also uncommon. Another
interesting feature of the S. capsulata aminopeptidase is
that, for an unknown reason, it is able to distinguish between similar
amino acid residues, like tyrosine and phenylalanine, in the case of
non-protected peptides (Table III).
Metalloaminopeptidases are predominant in bacteria (1). There are
several indirect indications that this is a zinc metalloenzyme. First,
effective inhibition of the S. capsulata aminopeptidase by
o-phenanthroline, but not by phenylmethylsulfonyl fluoride or p-chloromercuribenzoic acid, was observed. The enzyme is
also inhibited by certain anions, whose zinc salts have low solubility product value. We assume that EDTA, another strong chelator of transition metals, shows practically no inhibitory effect because of
its voluminous structure and polyanionic nature resulting in its
inability to penetrate close enough to the zinc of the catalytic site.
A neutral pH optimum (18) for the S. capsulata
aminopeptidase and a putative zinc binding domain HEXXH (15)
in its amino acid sequence are both indications of a zinc metalloenzyme.
Atomic absorption spectroscopy confirms the presence of one atom of
zinc per molecule of enzyme. In addition, four atoms of iron were also
detected. A plausible explanation might be nonspecific binding of this
metal to the protein molecule.
The results that have been presented prove the scientific novelty of
the S. capsulata aminopeptidase. We believe that this enzyme
will receive significant attention from the food industry, as well. It
has an "industrial" pH optimum, high specific activity, and great
performance in releasing alanine and glycine, two amino acids that give
considerably strong sweetness (19), in natural peptides.