From the § New England Biolabs Inc., Beverly,
Massachusetts 01915, the ¶ Department of Biochemistry and
Molecular Biology, College of Medicine, University of South Alabama,
Mobile, Alabama 36688, and the Boston Biomedical Institute,
Boston, Massachusetts 02114
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ABSTRACT |
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Glycosylasparaginase is an N-terminal nucleophile hydrolase and is activated by intramolecular autoproteolytic processing. This cis-autoproteolysis possesses unique kinetics characterized by a reversible N-O acyl rearrangement step in the processing. Arg-180 and Asp-183, involved in binding of the substrate in the mature enzyme, are also involved in binding of free amino acids in the partially formed substrate pocket on certain mutant precursors. This binding site is sequestered in the wild-type precursor. Binding of free amino acids on mutant precursors can either inhibit or accelerate their processing, depending on the individual mutants and amino acids. The polypeptide sequence at the processing site, which is highly conserved, adopts a special conformation. Asp-151 is essential for maintaining this conformation, possibly by anchoring its side chain into the partially formed substrate pocket through interaction with Arg-180. The reactive nucleophile Thr-152 is activated not only by deprotonation by His-150 but also by interaction with Thr-170, suggesting a His-Thr-Thr active triad for the autoproteolysis.
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INTRODUCTION |
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Glycosylasparaginase hydrolyzes the -N-glycosidic
bond between asparagine and N-acetylglucosamine of
asparagine-linked glycans (1). Glycosylasparaginases from different
sources consist of two non-identical
- and
-subunits held
together by strong non-covalent forces (2, 3). Glycosylasparaginase is
encoded by a single gene and is initially synthesized as a single
polypeptide that is post-translationally processed (2). This processing
is an obligatory step in the production of active enzyme, and cleavage of a single peptide bond is responsible for this activation (3, 4). The
newly formed N-terminal threonine residue of the
-subunit functions
as both the active base and nucleophile for the enzyme activity, which
places glycosylasparaginase in the N-terminal nucleophile-hydrolase
family (Ntn-enzyme)1
(5-7).
The three-dimensional structure of human glycosylasparaginase has been
published (8). The enzyme active center has been characterized and is
illustrated in Fig. 1A. The
proposed reaction mechanism is as follows. The hydroxyl of the
N-terminal nucleophile Thr-183 is probably activated by its own
-amino group and attacks the
-carbonyl of the asparagine part of
the substrate to form the tetrahedral transitional intermediate. The
negatively charged carbonyl oxygen of the transitional state is
stabilized by hydrogen bonds from the hydroxyl of Thr-234 and the main
chain nitrogen of Gly-235. The transitional intermediate collapses into
the acyl-enzyme intermediate and releases the sugar part of the
substrate. The acyl-enzyme intermediate is hydrolyzed by water. Besides
the interactions mentioned above, the substrate binding is achieved by
hydrogen bonding of the
-carboxylate and the
-amino group of the
substrate to Arg-211 and Asp-214, respectively. The hydrogen
interaction between Thr-183 and Thr-201 is also important for the
enzymatic activity.
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In a previous study (9), using a cloned glycosylasparaginase from Flavobacterium meningosepticum (10, 11), we have demonstrated that the activation is an intramolecular autoproteolytic processing event. Based on the experimental data, we proposed a cis-autoproteolysis model for glycosylasparaginase activation as illustrated in Fig. 1B. Since Ntn-enzymes have in common an unusual fold and the active N-terminal nucleophile is most often generated by autoproteolytic processing (5), we suggested that the cis-autoproteolysis model may also, with some variations, be held for processing of other Ntn-enzymes. Cis-autoproteolytic processing is a newly found post-translational modification that is involved in many important cellular processes, such as enzyme activation, proteasome biogenesis (12), hedgehog protein maturation (13), and protein splicing (14). A general catalytic mechanism of cis-autoproteolysis may be shared by many polypeptide main-chain modification pathways in living cells. Characterization and functional analysis of the autoprocessing active center in glycosylasparaginase should reveal the molecular mechanism of cis-autoproteolysis. This will provide valuable information for understanding these related cellular processing functions.
At present, the three-dimensional structure of glycosylasparaginase
precursor is not available. Identification and functional analysis of
the active residues for activation based on a three-dimensional structure of the precursor is not feasible. However, both the enzyme
reaction and the autoproteolysis resemble the reaction by
serine/cysteine proteases, and a single hydroxyl residue, Thr-152 in
the bacterial or Thr-183 in the human protein, is used as the reactive
nucleophile for both the reactions. Therefore, some other residues
involved in the enzymatic reaction may also be involved in the
autoproteolysis. On the other hand, the two reactions are chemically
different. The two active centers should also be different, both
structurally and functionally, although the two active centers may
share some part of their structures. For instance, the C-terminal residues of the -subunit are essential for activation but play no
roles in enzyme reaction. Therefore, in this study, not only were the
residues involved in the enzyme active center analyzed, but other
conserved residues that may be involved in the autoproteolysis were
also under investigation.
By studying in vitro autoprocessing of mutant precursors, we demonstrate that the activation of glycosylasparaginase follows the kinetics of cis-autoproteolysis. Site-directed mutagenesis and in vitro activation of purified precursors show that two residues, Arg-180 and Asp-183, which are involved in binding of substrate in the mature enzyme, are also involved in binding of free amino acids on mutant precursors. Binding of amino acid ligands on these precursors can either inhibit or stimulate their processing, depending on the individual mutants and amino acids. The results from this study suggest that Asp-151 plays an important role in activation similar to the P1 residue of substrates in protease reactions and that the partially formed substrate binding pocket on the precursor functions similarly to the S1 site of proteases. A charge interaction between Asp-151 and Arg-180 in the precursor is implicated. The data also suggest a possible His-Thr-Thr active triad in glycosylasparaginase activation.
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EXPERIMENTAL PROCEDURES |
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Materials-- The maltose-binding protein (MBP) protein fusion expression and purification system including plasmids pMAL-c2 and pMAL-p2, the Escherichia coli host strain TB1, amylose resin, Factor Xa protease, restriction enzymes, T4 DNA ligase, T4 DNA kinase, DNA polymerases, and synthesized oligonucleotides were obtained from New England Biolabs. The bacterial glycosylasparaginase clones used in this study were kindly provided by Dr. A. Tarentino (Wadsworth Laboratories, Albany, NY).
Enzyme Assay--
The glycosylasparaginase assay was based on
colorimetric measurement of N-acetylglucosamine released
from the substrate
N4-(-N-acetylglucosaminyl)-L-aspartic acid (AspNHGlcNAc) (Sigma) using the Reissig modification of the Morgan-Elson reaction as described previously (11).
Recombinant DNA and Mutagenesis-- DNA manipulation and site-directed mutagenesis (Kunkel methods) were carried out as described previously (9). All site-directed mutagenesis was performed using either the pMAL system or the Litmus system (New England Biolabs Inc.). DNA sequence analysis was carried out using an ABI automatic DNA sequencer.
Gene Expression and Protein Purification-- Expression and purification of gene products using the MBP fusion and expression system (15) with modifications were described previously (9), except that for expression of the glycine inhibition-minus mutants such as the T152S/D183N double mutant, a lower induction temperature (15 °C, overnight) or shorter induction time (3 h, 30 °C) was used.
In Vitro Autoproteolysis--
Amylose-purified fusion proteins
stored at 70 °C in the reaction buffer (20 mM Tris, pH
7.4, 50 mM NaCl, 1 mM EDTA) were thawed and
diluted in the same ice-cold buffer to the desired concentrations and
then shifted to an appropriate temperature to start autoproteolysis. At
various times, aliquots were withdrawn and subjected to SDS-PAGE
analysis. For some Thr to Cys mutant proteins, 5 mM
dithiothreitol was included in the reaction buffer to prevent oxidation
during long incubations. Protein gels were stained with Coomassie Blue
R-250, and protein bands were quantified by gel scanning using a
Microtek Scanmaker III, Adobe PhotoshopTM, and NIH Image 1.57.
Protein Analysis-- Protein concentration was determined by the Bio-Rad protein Assay using bovine serum albumin as standards. N-terminal protein sequence analysis was performed on an Applied Biosystems 610A as described previously (9). Molecular weight determination was carried out by SDS-PAGE analysis or electrospray mass spectrometry.
Kinetic Analysis-- The cis-autoproteolytic processing of the glycosylasparaginase proenzyme is described by the following reaction,
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(Eq. 1) |
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(Eq. 2) |
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(Eq. 3) |
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(Eq. 4) |
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(Eq. 5) |
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(Eq. 6) |
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(Eq. 7) |
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RESULTS |
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We fused the genes of the E. coli MBP and
glycosylasparaginase, termed MG, and expressed the gene fusion in
E. coli. The amylose-purified MG product consists of two
polypeptides. One contained a fusion between MBP and the -subunit,
termed MG
, with a molecular mass of 60 kDa. The second was the
glycosylasparaginase
-subunit with a molecular mass of 15 kDa. The
two polypeptides were in tight association, termed MG
, and
co-purified on amylose resin. Protein fusions between MBP and mutant
glycosylasparaginases were termed in the single-letter format for amino
acids as in the following example: MBP-glycosylasparaginase(T152A) was
termed MG(T152A) where 152 indicates the residue number based on
wild-type glycosylasparaginase. The precursor of MG(T152X) protein was
termed pre-MG(T152X), and the activated protein was termed
MG(T152X)
.
The Role of the N-O Shift in the Processing Reaction--
Our
previous study (9) showed that pre-MG(T152C) was processed very slowly
(the half activation time
48 h, 3 × 10
4
of the rate for wild-type) because a slowly hydrolyzed thioester intermediate was involved. This intermediate could be rapidly resolved
by adding the strong nucleophile hydroxylamine (NH2OH), which was highly reactive with an ester or a thioester. In the presence
of 0.25 M NH2OH, the half activation time
of pre-MG(T152C) was only about 30 min. When purified pre-MG(T152C) was
first denatured with SDS followed by incubation with NH2OH,
no precursor processing was observed (see
Fig. 2A, lanes 1 and 2). This indicated that no detectable thioester
intermediate existed in the protein sample before incubation. When
pre-MG(T152C) was first incubated for 3 h at 37 °C and then SDS
and NH2OH were added, followed by incubation for an
additional 1 h, the processing results were the same as in samples
treated identically for 3 h but without SDS and NH2OH. Only about 5% of the precursor was processed (lanes 3 and 4). When NH2OH was included in the reaction
during incubation, more than 95% of the precursor was processed after
3 h at 37 °C (lane 5). These results indicate that
although the thioester intermediate forms rather rapidly, no
accumulation of the hydrolysis-resistant intermediate takes place
during the autoproteolytic processing. Thus, the N-S shift step in the
autoproteolysis was reversible. His-150 is the proton acceptor/donor
for the autoproteolysis (9), and therefore activation of pre-MG(H150S)
is very slow (
5-7 days, compared with 1-2 min for pre-MG)
because of the very slow N-O shift rate. However, the processing rates
of pre-MG(H150S) could still be increased 3-5-fold when 0.25 M NH2OH was added (Fig. 2B). This
rate increase was similar to that of the fast processing pre-MG(T152S)
(
20 min without and
4 min with NH2OH).
This indicates that the hydrolysis step in activation of pre-MG(H150S)
is still the rate-limiting step. It cannot be reassigned from the ester
hydrolysis step to the N-O shift step simply by decreasing the N-O
shift rates. This mechanistically characterizes a reversible N-O shift
step being involved in the autoproteolysis (16) (also see
"Experimental Procedures").
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Characterization of the Amino Acid Binding Site on
Precursor--
The activation of pre-MG(T152S/C) is inhibited by small
-amino acids such as glycine and not by glycosylasparaginase
substrate (AspNHGlcNAc) or inhibitor (Aspartate) (9), indicating that the amino acid binding site on these precursors is different from the
substrate binding site of the mature enzyme. The location and
biological function of this amino acid binding site was unknown. To
address these questions, we undertook characterization of the residues
involved in binding of the amino acid ligands on the precursor.
Secondary mutations were introduced into the glycine-sensitive MG(T152S) mutant. We searched for suppressor mutants in which the
activation was no longer inhibited by glycine. Two residues, Arg-180
and Asp-183, were identified as the suppressor mutation sites
(Fig. 3). The freshly purified
MG(T152S/D183N) gene product consisted of about 20% precursor and 80%
processed protein. The half processing time
of pre-MG(T152S/D183N)
was about 40 min, similar to that of pre-MG(T152S) (
20 min).
The purified MG(T152S/R180Q) was mainly precursor, and the
half-processing time
of pre-MG(T152S/R180Q) was more than 72 h. The activation of both pre-MG(T152S/D183N) and pre-MG(T152S/R180Q)
was not inhibited by glycine, suggesting that Arg-180 and Asp-183 were
involved in amino acid binding on the precursor. The activation of
other double mutants such as MG(T152S/T170A), MG(T152S/T203A),
MG(T152S/T64A), MG(T152S/D58N), and MG(T152S/C68S) were still inhibited
by glycine. For example, the autoprocessing of pre-MG(T152S/T203A) was
severely inhibited by 10 mM glycine (Fig. 3), suggesting
that Thr-203 did not play significant roles in amino acid binding on
this precursor.
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Analysis of Aspartic Acid Mutants--
In serine proteases, the
active triad Ser-His-Asp forms a charge relay system to increase the
nucleophilic activity of the active serine and to stabilize the
transitional state of the reaction (17). To investigate whether
glycosylasparaginase activation involves a similar charge relay system,
we compared the sequences of six glycosylasparaginases from different
sources (7) and identified seven conserved aspartate residues; Asp-33,
-58, -66, -71, -151, -183, and -194. Each of these seven aspartate
residues was individually replaced with asparagine by site-directed
mutagenesis. The freshly purified MG(D33N), MG(D58N), MG(D66N),
MG(D71N), and MG(D194N) were present mainly as the processed
/
-subunit form with 0-10% of the protein presented as
precursors (Fig. 4A). The precursors were completely processed after incubation for 2-3 h at
37 °C (Fig. 4B). These results suggest that Asp-33, -58, -66, -71, and -194 do not play significant roles in the activation. For
MG(D183N), 70% of the purified protein was processed and 30% existed
as pre-MG(D183N). The processing rate of pre-MG(D183N) was about
100-fold reduced (
2 h) relative to the wild-type. Purified
MG(D183E) was already processed to MG(D183E)
. The enzymatic activities of MG(D183N/E)
were greatly reduced. Pre-MG(D151N) was
not processed. Asp-151 was further replaced with different residues by
site-directed mutagenesis using a synthesized DNA oligo-mixture where
codon 151 was randomized. Thirteen mutations were isolated;
Asp-151-Ala/Leu/Val/Met/Pro/Gly/Gln/Asn/Ser/Thr/Arg/His/Glu. All the
mutant gene products were purified as precursors. Purified MG(D151E)
existed mainly as pre-MG(D151E). The processing rate of pre-MG(D151E)
was decreased about 1000-fold (
20 h). Pre-MG(D151G) was
processed very slowly. Since MG(D151E/G)
was fully active, we
were able to estimate the processing rate of pre-MG(D151G) by measuring
the increase in enzyme activity. The processing rate was about 3 × 10
5 of the wild-type. All other mutants were inactive
in autoprocessing. Taken together, these results indicate that Asp-151
is virtually required for activation but not for enzyme activity.
Asp-183 is important for enzyme activity but not for activation.
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Isolation of Mutants with Amino Acid-sensitive Activation--
The
activation in wild-type is not inhibited by free amino acids
(Ki of glycine 10 mM) suggests
that the amino acid binding site is covered or sequestered in the
wild-type precursor. To investigate the residues that are involved in
sequestering the amino acid binding site, we set out to isolate mutants
whose processing rates were influenced by exogenous amino acids.
Several dozen single and double mutants at 22 residues, including 16 conserved ones (Asp-33, -58, -66, -71, -107, -151, -183, and -197;
His-150; Thr-64, -152, -170, and -203; Arg-180; Trp-11; Glu-97, and
-121; Cys-68, -81, -168, -232, and -270) were constructed, and the gene products were analyzed. Among all of the characterized mutants, only in
those mutated at His-150 or Thr-152 was the activation of the gene
products inhibited by glycine (data not shown). For example, the
activation of pre-MG(T152S/C) or pre-MG(H150S/K/W) was almost
completely prevented by 10 mM glycine, although some of
precursors were processed very slowly (Fig. 2B). The
glycine-sensitivity of activation in MG(H150X) mutants was irrelevant
to the size, charge, hydropathy, and geometry of the replacement
residues.
Stimulation of Autoproteolysis by Glycine-- The low processing rate of pre-MG(D151G) increased only slightly (2-3-fold) in the presence of 0.25 M NH2OH, indicating that a slow N-O shift step involved in the processing (see "Experimental Procedues"). Sterochemically, it is unlikely for Asp-151 to form a charge relay system with His-150 and Thr-152 because of the physical position of Asp-151. A possible role for Asp-151 is to anchor its side chain into a pocket through charge interaction to produce the correct conformation for the processing site polypeptide, i.e. Asp-151 functions similarly to the P1 residue of protease substrates. Arg-180 in the amino acid binding site of the precursor would provide an ideal site for binding Asp-151 (see Fig. 1A). In this case, one would anticipate that the activation of pre-MG(D151G), where the acetic acid side chain of Asp-151 is replaced by a hydrogen atom, may not be inhibited by glycine since the binding pocket could fully contain the bound glycine without interfering with the processing site folding. Indeed, the processing rates of pre-MG(D151G) were actually increased about 2-fold when 10 mM glycine was presented (Fig. 5A), although the processing rates were low. As expected, other amino acids could still inhibit the autoproteolysis to various degrees since their side chains could interfere with the processing site peptide folding over the binding pocket. The processing rates were reduced 15- to 20-fold with 10 mM threonine, aspartic acid, or histidine relative to that without free amino acids. The reduction was only 50% or less with 10 mM alanine, leucine, tyrosine, or lysine (Fig. 5B). Autoprocessing of pre-MG(D151G) was slightly stimulated by 0.2 M sodium acetate (Fig. 5B).
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Analysis of Threonine Mutants--
We have shown that the reactive
nucleophile Thr-152 for enzyme activity is also the reactive
nucleophile for autoproteolysis (9). Two other conserved threonine
residues in the enzyme active center, Thr-170 and Thr-203 (see Fig.
1A), were also investigated for their possible function in
the activation. The purified MG(T203S) was already processed to
MG(T203S), which retained 50% of the enzyme activity. The
processing rate of pre-MG(T203A) was about 100-200-fold reduced (
3 h) relative to the wild type. MG(T203A)
was about 30%
active. These results suggest that Thr-203 may not play essential roles
either in the precursor activation or in the enzyme activity. Thr-170
was replaced with Ser, Cys, and Ala, respectively, and the activation
of each mutant gene product was analyzed. The processing rate of
pre-MG(T170S) was reduced 10-20-fold relative to the wild-type. The
processing rates of pre-MG(T170A/C) were reduced about 1000-fold. To
understand these profound effects of Thr-170 on activation, we
investigated the possible interaction between Thr-170 and the reactive
nucleophile Thr-152 in the precursor since x-ray analysis showed that
the two corresponding residues, Thr-201 and Thr-183, in the human
enzyme were in hydrogen bond interaction (see Fig. 1A).
Single and double mutants were constructed and analyzed. The results
are summarized in Table I. These results showed the following. (a) The processing rates in the
mutants that contained both hydroxyl residues at position 152 and 170 were considerably faster than in the mutants that lacked hydroxyl residues at one or both of these positions. (b) Comparing
the k' values, which indicated the N-O or N-S shift rates
(see "Experimental Procedures"), among the different mutants showed that the lower processing rates in T170A/C single or in T152S/T170A/C double mutants were due to lower N-O shift rates since the relative hydrolysis rates (k/k') for these mutants were
actually the same (0.2-0.3) (T170A was an exception, the
k/k' = 0.04). (c) The processing rates
in T152C and T152C/T170A/C/S were slow but could be greatly accelerated
by adding NH2OH (k/k' = 0.01-0.03).
In contrast to N-O shifts in mutants with hydroxyl 152, the N-S shift
rates in mutants with thiol 152 became independent of the presence of a
hydroxyl group on residue 170. As a whole, the results showed that the
presence of both hydroxyl residues 152 and 170 was required for the
efficient activation and maintenance of the enzyme activity.
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DISCUSSION |
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The experimental data from in vitro activation of glycosylasparaginase indicate that activation of glycosylasparaginase is an intramolecular autoproteolytic event (9), and the experimental results from analysis of the mammalian 20 S proteasome biogenesis are consistent with a cis-autoproteolysis model (12). On the other hand, a study on autocatalytic processing of the 20 S proteasome from Thermoplasma acidophilum suggests that this processing is probably intermolecular (18). Therefore, more studies on the autoprocessing mechanism in different Ntn-enzymes are needed to determine if there is a common one for their activation. In this study we characterized the autoprocessing catalytic center of glycosylasparaginase. The results provided further evidence for cis-autoproteolytic processing of this Ntn-hydrolase.
Two types of intramolecular autoproteolysis have been reported. In the
first type, the active hydroxyl or thiol residue attacks a distant
peptide carbonyl on the same polypeptide to initiate autoproteolytic
processing. Autocleavage of LexA repressor is an example of this type
of reaction. This intramolecular processing has been successfully
converted to an intermolecular one by genetic manipulation (19, 20). In
the second type, or so-called cis-autoproteolysis, the active
nucleophile attacks the carbonyl at its N-terminal side (1 carbonyl)
to start the processing. Activation of glycosylasparaginase is such a
cis-autoproteolytic processing. Cis-autoproteolytic processing is not
enzymatic but instead is a catalyzed chemical reaction. In contrast to
proteolysis by serine/cysteine proteases, in cis-autoproteolysis,
formation of the ester or thioester intermediate via an N-O or N-S
shift is a reversible step. The released amino group cannot diffuse
from the catalytic center and therefore is ready to attack the ester
carbonyl to restore the peptide linkage. The equilibrium is favorable
for peptide bond formation via an O-N or S-N shift (21, 22), and
therefore no significant amounts of ester or thioester intermediates
are accumulated in slow ester hydrolysis mutants. The overall rate of
cis-autoproteolysis is determined not only by the N-O or N-S shift rate
but also by the O-N or S-N reverse shift rate and the hydrolysis rate
of the ester intermediate. The hydrolysis of the ester intermediate is
the rate-limiting step. The overall processing follows first-order kinetics. The transitional tetrahedral intermediate engaged by cis-autoproteolysis is shared by many post-translational modification pathways in living cells such as autocleavage (23), autoproteolysis (13), protein splicing (14), and formation of oxazole and thiazole
structures (24).
In this study, two residues, Arg-180 and Asp-183, were identified as
being involved in binding of amino acids such as glycine to certain
mutant precursors. The enzyme Km values in R180Q and
D183N mutants were greatly increased (27). The three-dimensional structure analysis showed that two corresponding residues, Arg-211 and
Asp-214, in human glycosylasparaginase were involved in binding of
the -carboxyl and the
-amino group, respectively, of the aspartic
acid as product co-crystallized in the enzyme active center (8) (see
Fig. 1A). Thus, our original suggestion that the amino acid
binding site found on the precursor is the partially formed substrate
binding pocket for the enzyme has now been proven (9).
Mutations at His-150/Asp-151/Thr-152 were able to produce amino acid-sensitive mutants (Figs. 2B, 3, and 5), suggesting that this processing site tripeptide is involved in sequestering the amino acid binding site on the wild-type precursor. Mutations at His-150, disregarding the size, charge, hydropathy, and geometry of the replacement residues (Ser, Lys, or Trp), produced the same glycine-sensitive gene products, suggesting that His-150 may specifically interact with another residue, most likely Thr-152, to prevent free amino acids from access to the binding site rather than directly interfering with the amino acid binding. This is also consistent with the fact that His-150/Thr-152 is the active base/nucleophile pair for autoproteolysis.
For a hydroxyl or thiol side chain to attack the adjacent 1 carbonyl
carbon, certain Ramachandran angles around the cleavage site dipeptide
are required. Replacement of Asp-151 with a non-acidic residue other
than glycine resulted in an inactive precursor because an incorrectly
folded processing site may result. Pre-MG(D151G) could slowly undergo
the autoproteolysis because a side chain-free residue, Gly-151, may fit
into the correct processing site conformation with less free energy
costs than other non-acidic residues. This is consistent with the fact
that glycine residues are frequently found at
1 position in many
similar bioprocessing systems (12-14, 24).
Among the seven conserved aspartate residues of
glycosylasparaginase, only mutations at Asp-151 produced profound
effects on activation (Fig. 4). The experimental data suggest that
Asp-151 most likely plays a role in the activation similarly to the P1 residue of substrate in the reaction by trypsin, where the interaction between the P1 residue of substrate and the S1 site of the enzyme is
the primary determinant in the enzyme-substrate binding. We proposed
that Asp-151 side chain was anchored into the partially formed
substrate pocket (the S1 site) by interacting with Arg-180. Possible
interaction between the 1 residue and the substrate pocket was also
suggested in an attempt to account for proenzyme activation in
Ntn-hydrolases solely based on their unusual folding (5). But the
experimental evidence was lacking. On the other hand, based on our
hypothesis, the molecular mechanism for inhibition of the activation by
amino acids can be well explained. Binding of an amino acid would
interrupt the interaction between Asp-151 and Arg-180 and displace the
side chain of Asp-151 from the pocket, and this would introduce
conformation change and abolish the autoproteolysis. This hypothesis is
further supported by the following facts. 1) Mutations at Arg-180
caused significant reductions in the processing rates and often
resulted in misfolded precursors (25, 27), while mutations at Asp-183
did not profoundly affect the activation. 2) The partially formed
substrate pocket on the wild precursor was sequestered by the
processing site His-150/Asp-151/Thr-152 tripeptide. 3) Asp-151 was
virtually required for activation. 4) Instead of inhibition of the
activation, glycine actually stimulated the activation of pre-MG(D151G)
(Fig. 5), which is very similar to the observation that methylamine
inhibits trypsin hydrolysis of specific substrates and stimulates the
enzyme activity toward nonspecific substrates (26). This also suggested
that the charge interaction between Asp-151 and Arg-180 had a role in
keeping the correct conformation of the precursor. As predicted, other amino acids were still able to inhibit the activation of pre-MG(D151G) as long as they could access the binding site.
The experimental data showed that the N-S shift rates of pre-MG(T152C) and pre-MG(T152C/T170A/C) were similar (Table I), which indicated that hydroxyl 170 was unlikely to take part in stabilization of the oxyanion at the transitional state of autoproteolysis. Thus, the fact that the N-O shift rates in the mutants with hydroxyl residue 170 are significantly higher than in the mutants with non-hydroxyl residue 170 may suggest that hydrogen bonding between the two hydroxyl residues 152 and 170 also exists in the precursor and plays an important role to increase the nucleophilic activity of Thr-152. The N-S shift rates in the mutants with thiol residue 152 were independent of hydroxyl 170 because thiol group hydrogen bonds poorly.
Tacking all available experimental data together, we proposed a model for the interactions of functional side chains involved in glycosylasparaginase activation as illustrated in Fig. 6. In a properly folded precursor, the polypeptide chain at the processing site adopts a specific conformation that makes possible the functional interactions between active side chains and allows the occurrence of an efficient N-O shift between Asp-151 and Thr-152. Thr-152 is activated not only by deprotonation by His-150, but may also by interaction with Thr-170. The side chain of Asp-151 is anchored into the partially formed substrate pocket, possibly through a charge interaction with Arg-180. This interaction helps to stabilize the processing site conformation. Arg-180 and Asp-183 in the partially formed substrate pocket are involved in binding of free amino acids to sensitive mutant precursors. Binding an amino acid to this site will displace the Asp-151 side chain from the pocket and thereby block autoproteolysis. In the wild-type precursor, interaction between His-150 and Thr-152 prevents free amino acids from binding to this site. Because formation of the ester intermediate via an N-O shift is a reversible reaction, with the equilibrium favoring the peptide bond formation, an efficient ester hydrolysis step is essential for this cis-autoproteolysis.
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Glycosylasparaginase activation provides a unique example that one catalytic center can be converted to another with different activity in a single protein through main chain modification and conformation change. X-ray three-dimensional structure analysis on the properly folded precursor should be able to reveal the detail conformation changes in this conversion.
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ACKNOWLEDGEMENTS |
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We thank Dr. Donald Comb (New England Biolabs) for supporting this research and Dr. Richard Roberts (New England Biolabs) and Dr. Nathan Aronson (University of South Alabama) for critical reading of the manuscript. We also thank Dr. P. Riggs and Dr. W. Jack for helpful discussions.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 978-927-5054;
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** Present address: Gryphon, 250 E. Grand Ave., South San Francisco, CA 94080.
1
The abbreviations used are: Ntn-enzyme,
N-terminal nucleophile-hydrolase; AspNHGlcNAc,
N4-(-N-acetylglucosaminyl)-L-aspartic acid; MBP, maltose-binding protein; PAGE, polyacrylamide gel
electrophoresis.
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