From the Department of Biochemistry and Molecular
Biology, University of South Alabama, Mobile, Alabama 36688 and
§ New England Biolabs, Inc., Beverly, Massachusetts
01915
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ABSTRACT |
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Flavobacterium glycosylasparaginase
was cloned in an Escherichia coli expression system.
Site-directed mutagenesis was performed at residues suggested to be
important in the catalytic mechanism based on the crystal structure of
the human enzyme and other biochemical studies. In vitro
autoproteolysis allowed the mutant enzymes to be activated, including
those that were slow to self-cleave. Based on the activity of the
mutant enzymes, six catalytically essential amino acids were
identified: Trp-11, Asp-66, Thr-152, Thr-170, Arg-180, and Asp-183.
Kinetic analysis of each mutant further defined the function of these
residues in substrate specificity and reaction rate. Mutagenesis of the
N-terminal nucleophile residue Thr-152 confirmed the key function of
its side-chain hydroxyl group. Partial activities of mutants T152S/C
were in agreement with the general mechanism of N-terminal nucleophile
(Ntn)-amidohydrolases. The side-chain hydroxyl of Thr-170 contributes
to the reaction rate based on studies of mutants T170S/C/A. Residues
Asp-183 and Arg-180 were found to H-bond, respectively, with the
charged -amino and
-carboxyl group of the substrate (Asn-GlcNAc).
Mutants R180Q/L and D183E/N had greatly decreased substrate affinity
and reduced reaction rates. Kinetic studies also showed that Trp-11 is
involved in regulation of the enzyme reaction rate, contradictory to a previous suggestion that this residue is involved in substrate binding.
Asp-66 is a new residue found to be important in enzyme activity. The
overall active site structure involving these catalytic residues
resembles the glutaminase domain of glucosamine 6-phosphate synthase,
another member of the Ntn-amidohydrolase family of enzymes.
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INTRODUCTION |
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Glycosylasparaginase (glycoasparaginase,
N4-(-N-acetyl-D-glucosaminyl)-L-asparagine
amidohydrolase) is a widely distributed amidohydrolase involved in the
ordered degradation of N-linked glycoproteins. It cleaves
the Asn-GlcNAc linkage that joins the oligosaccharide to the protein
(1, 2), and genetic deficiency of this enzyme causes the human
lysosomal storage disease aspartylglycosaminuria (2, 3). Reaction of
glycosylasparaginase with the linkage unit Asn-GlcNAc produces
aspartate and 1-amino-GlcNAc, which is further hydrolyzed
nonenzymatically to ammonia and GlcNAc (4). Previous studies have shown
the enzyme has a strict substrate specificity that requires both a free
-amino and
-carboxyl group on the asparagine residue. In
contrast, relatively flexible substrate requirements exist at the sugar
portion of the substrate. Substrates having mono-, di-, or even longer
saccharides are effectively hydrolyzed although the presence of
-L-fucose on the 6-hydroxyl of the
N-acetylglucosamine prevents the enzyme reaction (5-7). A
recent report suggests that the
-amino and
-carboxyl group of the
aspartate component of the substrate may not be strictly required for
enzyme activity (8).
Glycosylasparaginases have been purified or cloned from seven
biological sources, and all of them are encoded by a single gene
(9-11). After translation, the nascent single polypeptide is cleaved
into two nonidentical and
subunits by an autoproteolytic procedure (12). This self-cleavage produces the catalytically active
glycosylasparaginase heterodimer (13, 14). Glycosylasparaginases from
all the different species show a high sequence homology and conservation of biochemical properties (9, 15), and they are expected
to share a similar three-dimensional structure. Human glycosylasparaginase is folded into four layers of secondary structure,
-helices/
-sheet/
-sheet/
-helices (16), and shares unusual structural features of a recently recognized enzyme superfamily of
amidases, the N-terminal nucleophile
(Ntn)1 amidohydrolases (17,
18). The open cleft between the two central layers of antiparallel
-sheets is the position that incorporates the active site of these
enzymes (18). This protein fold is present in penicillin acylase (PA)
(19), the proteasome (PRO) (20), the glutaminase domain of glutamine
phosphoribosylpyrophosphate amidotransferase (GAT) (21), and
glucosamine 6-phosphate synthase (GLMS) (22), and it likely occurs in
other predicted family members. These enzymes are expected to manifest
a common catalytic mechanism. First, a unique property of Ntn
hydrolases is that the N-terminal amino acid acts as the reaction
nucleophile: O
of SerB1 in PA; S
of Cys-1 of GAT and GLMS (17,
18). Second, the free
-amino group of these same N-terminal residues
serves as a base to enhance the nucleophilicity of their side chain
hydroxyl or thiol group. The x-ray structure of human
glycosylasparaginase revealed not only its active-site N-terminal Thr
nucleophile/base but also other amino acids likely involved in
catalysis because the reaction product aspartic acid remained bound
within the crystallized protein (16).
In regards to the proposed catalytic groups of human glycosylasparaginase, some mutagenesis and expression experiments have already been done (24); however, limited kinetic data were obtained from these studies. A major obstacle was the inability to obtain pure autoproteolyzed mutant human enzyme in sufficient quantity to distinguish between the reaction mechanism for substrate hydrolysis and the autoproteolysis required to activate the single-chain precursor. Thus, it has been noted that many prospective active site residues are also likely to be involved in enzyme autoproteolysis, since precursor and mature enzyme may share a similar structure and the enzymatic mechanisms used in these two processes may resemble one another (17, 18). In this paper, we have applied a recombinant bacterial glycosylasparaginase system previously used by Guan (12) to mutate the predicted active site amino acids as well as other residues structurally close to the catalytic core. In vitro autoproteolysis and biochemical characterization of these mutants make it possible to investigate in detail the residues that are essential for precursor polypeptide folding, autoproteolytic activation, structural stability, and enzyme catalysis. Three different substrates were used to kinetically analyze the activity of these mutant forms of the enzyme, and the effects of these particular amino acid residues on either substrate specificity or glycosylasparaginase reaction rate have been measured.
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MATERIALS AND METHODS |
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Site-directed Mutagenesis of Recombinant Flavobacterium
Glycosylasparaginase--
Site-directed mutagenesis (Kunkel method)
was carried out as described by Guan (12). Wild-type
Flavobacterium glycosylasparaginase coding sequence (11)
was cloned into pMAL-p2 (New England Biolabs) as a maltose-binding
protein (MBP) chimera. Single-stranded phagemid that harbored wild-type
sequence was rescued by adding M13K07 helper phage (New England
Biolabs) to liquid-cultured transformed TB1 cells and was then isolated
by 20% polyethylene glycol, 2.5 M NaCl precipitation
followed by phenol/chloroform extraction, twice each. This
single-stranded phagemid was used as the template to conduct further
mutagenesis experiments. Primers with one to three base changes were
constructed to substitute single amino acids at the desired position.
Vectors bearing the designed substitution were selected based on
positional DNA sequencing of the mutation. Subsequent sequencing of the
entire protein coding region revealed no other mutations. At least
three independent clones of each mutant were picked. Proteins were
expressed in the TB1 cells by adding 1 mM
isopropyl-1-thio--D-galactopyranoside for induction at
30 °C overnight. Cells were pelleted by centrifugation, resuspended in 20 mM Tris buffer, pH 7.5, containing 100 mM
NaCl, and lysed by sonication. Enzymes were affinity purified over an
amylose column according to the protocol of the manufacturer (New
England Biolabs). After purification, the chimeric
glycosylasparaginases were digested with Factor Xa at 10 °C for 2-3
h. Cleaved MBP was removed by running the protease reaction through a
high-Q-Sepharose column (Amersham Pharmacia Biotech). All purification
procedures were performed at 4 °C. Protein expression efficiency
using this system was about 40 mg glycosylasparaginase in a 1-liter
culture.
In Vitro Autoproteolysis and Quantitation of Mutant
Glycosylasparaginase--
The protocol used to perform in
vitro autoproteolysis was done according to Guan (12). All steps
were done in succession immediately after affinity purification and
removal of MBP. Purified mutant glycosylasparaginases were concentrated
by Centricon 10 ultrafiltration (Amicon Corp.) and were immediately
analyzed on SDS-PAGE. Wild-type enzyme appeared as its separated and
subunits having molecular weights of approximately 15 and 18 kDa. Slow cleavage precursors required incubation at 37 °C for an
appropriate time. Autoproteolysis of these precursors was monitored by
taking aliquots at different time intervals and analyzing them on
SDS-PAGE. For the slowest cleavage mutants, an intermediate level of
formed glycosylasparaginase subunits was used to measure the amount of active enzyme. The cleaved forms were quantitated after SDS-PAGE and
Coomassie Blue staining by determining their image density using the
computer program NIH Image
1.62.2
Enzyme Assay and Determination of Kinetic Parameters Km and kcat-- Three different substrates were used to assay the activity of wild-type and mutant glycosylasparaginases, and the kinetic parameters Km and kcat were determined for each reaction. Due to very low activities of some enzymes and solubility limitations of some substrates, not all mutant enzymes were tested with all three substrates.
Asn-GlcNAc As Substrate Measured by Release of GlcNAc-- The standard reaction was 2.5 mM substrate in 20 µl of 20 mM sodium phosphate buffer, pH 7.5, incubated with enzyme for an appropriate time at 37 °C (25). The reaction was stopped by adding 50 µl of 250 mM sodium borate buffer, pH 8.8, followed by boiling for 3 min. Released N-acetylglucosamine was assayed by the Morgan-Elson reaction (26). The Km and kcat values of mutants R180K/Q/L and D183E/N were determined by this method. Experiments were performed by incubating 12-15 different concentrations of Asn-GlcNAc from 1 to 20 mM with R180K, 1 to 50 mM with D183E, and 10 to 100 mM with all other mutants. Substrate concentrations higher than 100 mM were also tried; however, less accurate results were obtained due to the level of free GlcNAc in this amount of substrate. Initial rates of GlcNAc release were measured when less than 5% of total substrate was hydrolyzed, and these values were used to determine Km and kcat.
Asn-GlcNAc and Asparagine As Substrates Measured by the Release
of Aspartate--
The above traditional colorimetric method measuring
GlcNAc has an assay limit at about 0.1 mM which was not
sensitive enough to do a precise kinetic study of many of the mutant
enzyme forms. Therefore, enzyme activity was also assayed by a coupled
enzymatic procedure originally described by Tarentino and Maley (27)
and based on: 1) the hydrolysis of the two substrates by
glycosylasparaginase with the release of aspartate; 2) the subsequent
transamination of aspartate to oxaloacetate by glutamate-oxaloacetate
transaminase (GOT) in the presence of -ketoglutarate; and 3)
formation of NAD+ after malate dehydrogenase (MD) reduction
of oxaloacetate to malate with NADH. Formation of NAD+ was
then measured fluorometrically using a Perkin-Elmer LS50B Luminescence
Spectrometer with the excitation wavelength at 360 nm and emission at
460 nm. Each reaction was performed in 150 µl of 20 mM
sodium phosphate, pH 7.5, 0.1 mM
-ketoglutarate, 0.2 mg
of NADH, 7.5 units each of GOT and MD with a glycosylasparaginase substrate and an appropriate amount of wild-type or mutant enzyme. All
reagents and enzymes for the GOT and MD steps were obtained from Sigma.
The assay mixture was incubated at 37 °C, and the reaction was
terminated by vigorously mixing with 20 µl of 6 N HCl to
destroy excess NADH followed by adding 100 µl of 10 N
NaOH (28). Samples of 200 µl from each reaction were placed in a 96-well plate to measure the fluorescence of the NAD+ final
product. Sensitivity of this assay allows measurement of 0.002 mM NAD+, which enabled accurate determination
of enzyme hydrolysis over a wide range. To determine the
Km and kcat of enzyme hydrolysis, 10-15 concentrations of substrate were used, generally ranging from 0.2 to 5 times Km. Initial rates were
calculated by terminating reactions at three to five different time
points. The highest substrate concentration used was 30 mM
for Asn-GlcNAc and 8 mM for asparagine. Results were not
accurate at higher substrate concentrations by this method because of a
high background from contaminating free aspartate.
Aspartic Acid -(p-nitroanilide) (Asp(pNA)-OH) As Substrate
Measured by p-Nitroaniline Release--
Each reaction was in a volume
of 200 µl of 50 mM Tris buffer, pH 7.5, containing
appropriate concentrations of aspartic acid
-(p-nitroanilide) (Bachem) substrate and wild-type or
mutant enzyme. Release of p-nitroaniline was monitored at
400 nM with a 37 °C temperature-controlled Beckman DU640
spectrophotometer (29). The Km and
kcat were determined by measuring the initial
hydrolysis rates at 12-15 concentrations of substrate ranging from 0.2 to 5 times Km. Because of the limiting solubility of
Asp(pNA)-OH in water, the highest concentration of substrate used in
this assay was 8 mM. The maximum reaction time used for
Asp(pNA)-OH and Asn substrates was not over 1 h, and nonenzymatic
hydrolysis did not occur during this period of incubation at 37 °C.
Asn-GlcNAc was assayed for 3 days with the T152C and T170A mutant
enzymes and was stable by itself for this long incubation.
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RESULTS |
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Generation of Active-site Mutants, in Vitro Autoproteolysis, and
Activity Analysis--
Residues of Flavobacterium
glycosylasparaginase selected for mutagenesis (Table
I) were mostly the counterparts of
essential human glycosylasparaginase residues (16, 30). The expression and purification of wild-type enzyme are shown in Fig.
1a (lanes 1-5).
The same protocol was used to express and purify mutant enzymes. No
obvious different behavior was observed for these mutants during the
procedures, and all recombinants were expressed as stable proteins. The
wild-type banding pattern was observed for mutants S50A, D58N, T64A,
D107N, T169A, K176A, and T203S, indicating these mutations did not
interfere with polypeptide folding or their autoproteolytic activation.
All other mutagenesis of the targeted amino acids resulted in
incomplete or poor initial autoproteolysis with an uncleaved precursor
band () present at about 32 kDa. Incubation of these mutants at
37 °C allowed most of them to be cleaved into their subunits (Fig.
1b; Table I). In the case of the R180K mutant, the majority
of its precursor precipitated during the 37 °C incubation (Fig.
1c). The same phenomenon occurred with all other R180
mutants (data not shown). Mutants W11F and T152A did not undergo
autoproteolysis (Fig. 1b). Activity of wild-type enzyme and
each mutant was assayed colorimetrically using 2.5 mM
Asn-GlcNAc as substrate after the mutant precursors had completed their
autoproteolysis (Table I). Almost all mutant enzymes that were
"slow" to form subunits (T1/2 > 60 min) were
poorly active once autoproteolysis took place. The T203A
glycosylasparaginase (T1/2 = 3.5 h) was an
exception and retained substantial catalytic capacity (31% of
wild-type enzyme).
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Temperature Stability of Wild-type Glycosylasparaginase and Mutants-- Glycosylasparaginases from most organisms are known to have high thermostability (15), but the Flavobacterium enzyme is noticeably less heat stable than the human enzyme (32). Therefore, the stability of the mutant recombinant enzymes was examined at different temperatures (Fig. 2). Wild-type enzyme began to inactivate at 55 °C and was completely inactivated at 60 °C. Mutants D107N and T169A began to inactivate about 5-10 degrees earlier than wild-type. Three other mutants, S50A, D58N, and T203A, were completely inactivated at 55 °C. D183N had an unstable structure and precipitated at 50 °C. D66N had the least stable structure and was barely active even at 40 °C.
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Kinetic Characterization of Wild-type and Mutant
Enzymes--
Glycosylasparaginase kinetic parameters
Km and kcat were determined
using the natural substrate Asn-GlcNAc or the alternate compounds,
asparagine or Asp(pNA)-OH (see "Materials and Methods"). Enzyme
residues that might interact with the oligosaccharide could be studied
by comparing hydrolysis of asparagine and Asp(pNA)-OH to that of
Asn-GlcNAc (Table II). A typical kinetic
analysis of the initial rates as a function of substrate concentration
is shown in Fig. 3. Wild-type enzyme had
a Km for the natural substrate Asn-GlcNAc of 0.085 mM, which is similar to human glycosylasparaginase (33).
The kcat was 16 s1. A contribution
of GlcNAc to the enzyme-substrate interaction and reaction was
indicated by a 7-fold increase of the Km and a
13-fold decrease of kcat when asparagine was
used as substrate. The nitroaniline analogue was a somewhat better
substrate than asparagine probably because the aromatic nitroaniline
group is a better leaving group and might also improve substrate-enzyme binding.
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Mutants T152S/C and T170S/C/A--
Substitution of the -subunit
N-terminal nucleophile Thr-152 by Ser reduced
kcat about 20-fold. Relative to wild-type
enzyme, the Km of T152S for the three tested
substrates, especially for asparagine or Asp(pNA)-OH, was much less
affected than kcat. Substitution of the
nucleophile hydroxyl by a thiol group (T152C) yielded an enzyme that
was barely active, and the only kinetic parameter measured was the
kcat (1.5 × 10
4
s
1) after overnight assay with saturating Asn-GlcNAc
substrate.
Mutants R180K/Q/L and D183E/N/A--
Mutation of Arg-180 or
Asp-183 reduced the catalytic reaction rate. These amino acids
correspond to residues in the human enzyme that H-bond respectively to
the -carboxyl and
-amino group of the substrate. Both
Km and kcat were adversely affected and the specificity constant
(kcat/Km) was decreased from
200 to 106-fold compared with wild-type enzyme. Mutant
R180K had an 8-fold increased Km for Asn-GlcNAc as a
substrate. Mutation to a Gln (R180Q) with a polar side-chain amide
caused a much greater increase in Km (200-fold).
Replacement with a nonpolar side-chain residue (R180L) further
increased the enzyme Km about 400-fold compared with
that of wild-type enzyme. Mutation of Arg-180 also reduced the enzyme
catalytic rate. The kcat values of R180K/Q/L
were at least 10-fold lower than that of wild-type glycosylasparaginase.
Mutants T203S/A--
From the crystal structure of human
glycosylasparaginase, it was predicted that the negative -carbonyl
oxygen of the Asn component of the substrate in its tetrahedral
transition state is stabilized by an oxyanion hole (16). The O
of
conserved Thr-203 and the main-chain nitrogen of Gly-204 were suggested to play major roles in this structure by hydrogen bonding to the carbonyl oxygen (16). However, mutagenesis of bacterial Thr-203 to Ser
or Ala did not substantially influence Km for any of
the three substrates (Table II). With Asn-GlcNAc as substrate, the
mutant T203A surprisingly showed a 50% decrease in
Km with only about 10-fold reduction of
kcat. The decrease in hydrolysis rate by this
mutant possibly results from slowing down the deacylation step
(k3) of the enzyme reaction (see
"Discussion"). The difference between reaction rates in having the
side-chain hydroxyl of residue 203 on Ser or Thr was small in the case
of Asn as substrate.
Mutants W11S and D66N-- Kinetic analysis of the bacterial mutant W11S showed reductions of enzyme specificity for substrates both with carbohydrate (Asn-GlcNAc) or without (asparagine or Asp(pNA)-OH). In addition, more significant changes occurred in kcat, which was reduced more than 400-fold for Asn-GlcNAc and relatively less for the other two substrates (20- and 65-fold, respectively).
Asp-66 was found to greatly affect enzyme activity. Kinetic characterization of a D66N mutant using the substrate Asn-GlcNAc revealed a l000-fold reduction in the specificity constant kcat/Km. This change reflects both an increase in the Km and a decrease in kcat. However, for this same mutant, kcat/Km was reduced only 12-fold when asparagine was used as substrate, which was due only to a reduction in kcat. In contrast, the Km of D66N for Asp(pNA)-OH increased by more than 20-fold compared with that of wild-type enzyme. ![]() |
DISCUSSION |
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In this study, we have used site-directed mutagenesis of a
bacterial form of glycosylasparaginase to separately examine the autoproteolytic activation and reaction mechanism of an important human
lysosomal enzyme that is also a member of the Ntn-amidohydrolase family
of proteins (17). Flavobacterium glycosylasparaginase was
originally isolated and cloned by the laboratory of Tarentino (11, 29),
and it has the simplest amino acid sequence among forms of the enzyme
from six different species, as it lacks about 30 amino acids at the C
terminus of its -subunit (9, 11). The same C-terminal region of the
human
-subunit is cleaved in lysosomes without any known effect on
the mature enzyme. The overall sequences of all known
glycosylasparaginases are highly conserved, especially the amino acids
predicted to be functionally essential based on the human enzyme
crystal structure and other studies (9, 13, 14, 16, 23, 30). Therefore,
the catalytic mechanism of the Flavobacterium enzyme
should generalize to other glycosylasparaginase members.
Maturation of glycosylasparaginase is an unusual process shared by many
members of the Ntn-amidohydrolase family. The nascent translated
precursor folds to a conformation that allows autoproteolysis of a
zymogen into and
subunits (12, 31). This step exposes the
N-terminal Thr of the
subunit to create the active site nucleophile
(T152 in this study) and possibly shift other important residues into
the final reaction center. Thr-152 is also the key catalytic amino acid
in autoproteolysis because it nucleophilically attacks the adjacent
Asp-151, allowing the peptide bond between them to be broken and an
active enzyme to be formed with a free
-amino group at the N
terminus of the
subunit (12, 17). Previous mutagenesis studies on
the human enzyme indicated that reductions of glycosylasparaginase
activity in many cases are due to a slowing down of precursor
autoproteolysis such that the enzyme is incompletely activated
(13, 14, 24). This complexity in glycosylasparaginase
processing creates experimental difficulties when investigating the
function of specific amino acids in the mechanism of enzyme
catalysis.
Flavobacterium glycosylasparaginase mutants W11S/F, E48A, D66N, T152S/C, T170S/C/A, R180K/Q/L/A, D183E/N/A, and T203A slowed precursor autoproteolysis to different levels (Fig. 1). Prolonged incubation at 37 °C in vitro allowed most of them to be cleaved and activated enabling us to analyze amino acids essential to the mechanism of this enzyme. Only W11F and T152A were not cleavable in this system (Fig. 1b). In contrast, human W11F was reported to be cleaved when transiently expressed in COS cells (23). Mutant T152C was also very slow to undergo autoproteolysis at 37 °C, and further increase in temperature did not accelerate cleavage but instead caused precursor precipitation (data not shown). Cleavage of T152C precursor can be accelerated by hydroxylamine (12). Residue Arg-180 appears to be important in precursor peptide folding since its mutation yielded unstable precursors (Fig. 1c) which aggregated at 37 °C. Decreasing the incubation temperature helped to prevent its precursors from precipitation and yielded a greater amount of activated enzyme (data not shown).
The reaction mechanism of Ntn-amidohydrolases resembles that of serine
proteases involving an enzyme acylation-deacylation (17, 19). Based on
its crystal structure, amino acid mutagenesis studies (13, 16, 17), and
molecular dynamics calculations (30), glycosylasparaginase has a
similar catalytic mechanism (Fig. 4).
N-terminal Thr-152 of the -subunit serves as the nucleophile which
is activated by its own free
-amino group. The O
of Thr-152 attacks the side-chain amide carbon of the substrate (Asn-GlcNAc). The
negatively charged carbonyl oxygen in the tetrahedral transition state
is then stabilized by an oxyanion hole. Collapse of this structure
releases the carbohydrate product and yields an aspartyl-enzyme intermediate. An entering water molecule nucleophilically attacks the
same carbon to form another tetrahedral transition state that collapses
to release the second product aspartate. The key residue in the
catalysis is Thr-152. Mutation of Thr-152 to Ser or Cys yielded much
less active mutant enzymes, 8% activity for T152S and <0.2% activity
for T152C (Table I). These results are consistent with the general
mechanism of Ntn-amidohydrolases. However, depending on the individual
Ntn-amidohydrolase, either Thr, Ser, or Cys functions as the
nucleophile. The large activity reduction for glycosylasparaginase
mutant T152S/C indicates that a precise chemistry of the nucleophile
must optimize each individual active site structure in this enzyme
superfamily. Residue Thr-170 has its side-chain hydroxyl closest to the
nucleophile O
of Thr-152. Previous studies suggested this hydroxyl
group forms a hydrogen bond that stabilizes the activated O
of
Thr-152 (16). Mutagenesis of bacterial Thr-170 to Cys and Ala greatly
affected the enzyme hydrolysis rate (Table II). Mutant T170C had a
reduction of the kcat with little effect on
Km compared with wild-type enzyme, which suggests this rate reduction is likely in the acylation step
(k2) (34, 35). Mutant T170A had a further
reduced value of kcat but was accompanied by a
decrease in Km which normally occurs when the
deacylation rate k3 is reduced
(Km=KS[k3/(k2+k3)]) (34, 35). Our study confirms that the side-chain hydroxyl of Thr-170 is
essential in maintaining the enzyme reaction rate, probably influencing
both acylation and deacylation steps.
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Hydrolysis initiates when the substrate is bound into the active site and binding is suggested to be achieved principally by side-chains of Arg-180 and Asp-183 and the main-chain carbonyl oxygen of Gly-204 which together form hydrogen bonds to the charged groups of the substrate (16). Mutagenesis of Arg-180 and Asp-183 greatly reduced enzyme specificity, which confirmed that interactions from these charged groups are crucial for substrate binding. However, these mutations also greatly affected the enzyme reaction rate, especially in the case of Asp-183. Mutant D183N reduced the kcat more than 1500-fold (Table II). Asp-183 has its negative side-chain close to Gly-204, and it also may be involved in forming the transition state oxyanion hole.
The role of an oxyanion hole is taken from the mechanism of serine proteases, where those amino acids interacting with the negatively charged carbonyl oxygen stabilize the tetrahedral transition state during peptide bond hydrolysis. In the case of subtilisin, preventing formation of hydrogen bonds in the oxyanion hole by mutating Asn to Ala reduces its reaction rate more than 1000-fold, which emphasizes the importance of this structure in hydrolysis (36). In contrast, the side-chain of Thr-203 of glycosylasparaginase did not appear to be as important in stabilizing the transition state as previously suggested (16). The values of kcat for T203S/A mutants decreased only about 5-10 fold (Table II). Whether a main-chain component of Thr-203 together with Gly-204 or another essential residue actually form the oxyanion hole structure thus remains unsettled.
The catalytically essential amino acids and their function within
glycosylasparaginase are comparable with those in another Ntn-amidohydrolase, the glutaminase domain of glucosamine 6-phosphate synthase (22). This domain catalyzes hydrolysis of glutamine to
glutamate and ammonia. Indeed, the reaction product glutamate was found
complexed in the crystal structure of this glutaminase (22), just as
aspartate was found in the catalytic core of human glycosylasparaginase
crystals (16). The active site of this glutaminase domain contains a
Cys-1 nucleophile, and its residues Arg-73 and Asp-123 respectively
hydrogen bond to the -carboxyl and
-amino group of the glutamate
product. The oxyanion hole of the glutaminase domain is formed by the
side-chain of Asn-98 and the main-chain NH of Gly-99. In addition,
there are two other important residues, Trp-74 and Asp-29. Trp-74 seems
to be involved in stabilizing the tetrahedral intermediate by
interaction with the amide nitrogen of the substrate. Asp-29 is
presumably involved in a "gate" mechanism that regulates the active
site in either an open or closed conformation (22). Mutagenesis of the
Flavobacterium glycosylasparaginase also reveals similarly
important residues Trp-11 and Asp-66. Our study contradicts the current
opinion that Trp-11 is involved principally in substrate binding due to
its interaction with carbohydrate (16, 23). Thus, our kinetic analysis
revealed that the aromatic side-chain of Trp-11 instead greatly
contributes to the enzyme reaction rate. Viewing the enzyme active-site
structure indicates this Trp locates outside of Thr-152 (Fig.
5), and it could function similarly to
Trp-74 in glutaminase. Mutant W11S reduced both Km
and kcat, suggesting this Trp is probably
involved in regulating the enzyme deacylation rate
k3 (Table II) (34, 35). Residue Asp-66 is
geometrically close to the side-chain of Arg-180, (Fig. 5) and it may
stabilize hydrogen bonding between the positively charged side-chain of Arg-180 and the
-carboxyl group of the substrate (Table II). How
this residue affects the reaction rate constant
kcat, how it affects the binding of the
nitroaniline substrate analogue, and whether glycosylasparaginase has a
similar gate conformation mechanism remains unclear. However, it was
noted that substitutions at both Trp-11 and Asp-66 affected enzyme
activity less when asparagine was used as substrate rather than
Asn-GlcNAc (Table II). Residue Asp-66 was also shown to be very
important in maintaining intact tertiary structure of the enzyme.
Changing Asp-66 to its amide Asn yielded an enzyme that rapidly
denatured when the temperature was over 37 °C, while wild-type
enzyme remains fully active almost to 55 °C (Fig. 2). Several other
amino acids (Ser-50, Asp-58, Asp-107, Thr-169, Asp-183, and Thr-203)
were also shown to contribute to enzyme thermostability.
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Residue Glu-38 is the counterpart of human glycosylasparaginase Asp-47
and is not conserved in the rat enzyme which has an Ala at this
position (9). Glu-48 together with two charged residues, Lys-176 (human
Lys-207) and Arg-180 (human Arg-211), were suggested by ab
initio quantum mechanics and molecular dynamics calculations to be
the most important amino acids for stabilizing the transition state
(30). However, the Flavobacterium mutant E48A showed an
increased enzyme activity of about 120% (Table I). Our study also
indicated that Lys-176 is not essential because mutant K176A retained
almost full activity (Table I). Ser-50 is the shortest distance from
O of nucleophile Thr-152 (30), and enzyme activity of the mutant
S50A was reduced to about 20% (Table I). This residue probably
stabilizes the nucleophile Thr-152 by hydrogen bonding with the
positive
-amino group (24), and yet based on the relatively small
change in enzyme activity, this role is not critical for catalysis.
Mutagenesis of two other Asp (Asp-58 and -107) and Thr (T64A and T169A)
residues that are structurally close to the active site only slightly
influenced enzyme activity (Table I).
Our results from site-directed mutagenesis of Flavobacterium glycosylasparaginase combined with the previous crystal structure (16) and biochemical (13, 14, 24) and theoretical studies (30) of the human enzyme identify the main catalytic residues at the active site of this Ntn-amidohydrolase to be Trp-11, Asp-66, Thr-152, Thr-170, Arg-180, and Asp-183 (Fig. 5). Still lacking is detailed information about enzyme specificity and mechanism, including residues involved in both acylation and deacylation steps and the subtle structural changes that occur during these parts of the reaction. Further three-dimensional structural information about wild-type and mutant enzymes is required to define the precise mechanism of this enzyme. As a model member of the very interesting Ntn-amidohydrolase superfamily, all biochemical aspects of glycosylasparaginases, from autoproteolytic activation to catalysis, are important to characterize and understand.
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ACKNOWLEDGEMENT |
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We thank Dr. C. Tao for providing mutation vectors T152S/C and for help with other laboratory techniques.
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FOOTNOTES |
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* This work was supported by Public Health Service Grant DK-33314 from the NIDDK, National Institutes of Health.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.: 334-460-6402; Fax: 334-460-6127.
1
The abbreviations used are: Ntn, N-terminal
nucleophile; PAGE, polyacrylamide gel electrophoresis: GOT,
glutamic/oxaloacetic transaminase; MD, malate dehydrogenase; MBP,
maltose-binding protein; PA, penicillin acylase; GAT, glutamine
phosphoribosylpyrophosphate amidotransferase; GLMS, glucosamine
6-phosphate synthase; Asp(pNA)-OH, aspartic acid
-(p-nitroanilide).
2 Available at rsb.info.nih.gov/nih-image/default.html.
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REFERENCES |
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