(Received for publication, August 23, 1995; and in revised form, November 7, 1995)
From the
The activation mechanism of glycosylasparaginase of Flavobacterium meningosepticum has been analyzed by site-directed mutagenesis and activation of purified precursors in vitro. Mutation of Thr-152 to Ser or Cys leads to gene products that are not activated in vivo but are activated in vitro because processing of the mutant precursors is inhibited by certain amino acids in the cell. Kinetic studies reveal that activation is an intramolecular autoproteolytic process. The involvement of His-150 and Thr/Ser/Cys-152 in activation suggests that autoproteolysis resembles proteolysis by serine/cysteine proteases. Multiple functions of the highly conserved active threonine residue are implicated.
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(4) . This processing is an
obligatory step in production of active enzyme and cleavage of a single
peptide bond is responsible for this
activation(5, 6, 7) . The newly formed
N-terminal threonine of the
-subunit plays an essential role in
enzyme activity(8) .
Aspartylglycosaminuria (AGU) ()(McKusick 20840), which is the most common and severe
disorder of glycoprotein degradation, is caused by a deficiency of
glycosylasparaginase(9) . The incidence of AGU is high in
Finland; in eastern Finland the frequency of defective gene carriers
was estimated to be about 1 in 40(10) . Most cases of AGU are
caused by a mutation in the gene that results in a failure to activate
the enzyme(7, 11) . A closely related
glycosylasparaginase occurs in Flavobacterium meningosepticum and its gene has been cloned into Escherichia coli. The
cloned enzyme was efficiently processed and fully
active(12, 13) . Since the bacterial enzyme and its
mammalian counterparts have the same enzymatic specificity, have
similar heterodimer structures, are activated in the same way,
cross-react with common antibodies, and share extensive similarities in
their polypeptide sequences(12, 13) , it is likely
that their molecular mechanisms of activation are identical. However,
little is known about the nature of the activation process. In this
study we have used the cloned bacterial enzyme to investigate the
mechanism of glycosylasparaginase activation.
For preparation of the nonfusion active mutant precursors of glycosylasparaginase, 10 mM glycine was included in all of the extraction and purification buffers to inhibit autoproteolysis. The amylose-purified fusion protein was digested with 2% Factor Xa in the presence of 10 mM glycine for 1 h at 37 °C. The nonfusion precursor was then separated from MBP and Factor Xa by FPLC with a Mono Q column. The glycine in the purified precursors was removed by dialysis against the reaction buffer (20 mM Tris, pH 7.6, 50 mM NaCl and 1 mM EDTA) with a Pierce Slide-A-Lyzer dialysis cassette, 4 changes in 60 min at 0 °C.
For testing inhibitors of
autoproteolysis, amylose-purified MG(T152S) protein was diluted to a
concentration of 5 µM in a reaction buffer containing an
inhibitor (amino acids) at different concentrations. The inhibition
constant K is determined by the
equation:
where [I] = the inhibitor concentration and
[E] = the enzyme concentration. Let
[I°] and [E°] equal the initial
inhibitor and enzyme concentration, respectively. For a first-order
reaction, the concentration of the inhibitor that doubles the half-life
of the pre-MG(T152S) equals the inhibition constant K.
Thus
If [I°] [E°], then K
[I°].
where H* is the activation enthalpy and
S* is the activation entropy of autoproteolysis. R is the gas constant, T is the absolute temperature, and
ln A is the frequency factor and can be taken as a constant
under the experimental conditions. Plotting ln k against
1/T (Arrhenius plots), the slopes of the linear plots provide
an estimate of the activation enthalpies -
(
H*/R).
where k and k
are the
rate constants of the wild type and the mutant fusion precursors.
S* is the difference of activation entropies of
autoproteolysis between the wild type and the mutant proteins.
We fused the genes of MBP and glycosylasparaginase and
expressed their product, termed MG, in E. coli(15) .
The amylose-purified MG consisted 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 normal
-subunit with
a mass of 15 kDa. The two polypeptides were in tight association,
termed MG
, and co-purified over an amylose resin. The rapid
maturation of glycosylasparaginase prevented isolation of properly
folded precursor.
Figure 1: SDS-PAGE analysis of glycosylasparaginase processing. Figure shows autoproteolysis of amylose-purified MG(T152X). 1, freshly purified MG(T152S); 2, MG(T152S) after incubation for 1 h at 37 °C; 3, freshly purified MG(T152C); 4, MG(T152C) after incubation for 36 h at 37 °C; 5, freshly purified MG(T152A); 6, MG(T152A) after incubation for 36 h at 37 °C. All reactions were in buffer containing 20 mM Tris, pH 7.6, 50 mM NaCl, 1 mM EDTA. SDS-PAGE was performed in a 4-20% precast gradient gel. The free MBP is the result of proteolysis in vivo and copurifies with the MBP-glycosylasparaginase fusion.
Figure 2:
Kinetic analysis of autoproteolysis of the
MBP-enzyme fusion precursor pre-MG(T152S). Amylose-purified MG(T152S)
protein was diluted to different concentrations in the reaction buffer
at 0 °C, then shifted to 37 °C to start processing. At various
times an aliquot of the protein sample was withdrawn and analyzed for
conversion of pre-MG(T152S) to MG(T152S). Pre-MG(T152S) at
concentrations of 0.1 mg/ml, 1 mg/ml or 10 mg/ml respectively,
separated in 4-20% gradient gels.
Figure 3: Kinetic analysis of autoproteolysis of the nonfusion precursor pre-G(T152S). Purified pre-G(T152S) was used for autoproteolysis, at a concentration of 0.5 mg/ml, and separated in a 10-20% gradient gel.
Figure 4: Glycine inhibition of autoproteolysis analyzed by SDS-PAGE. 1, freshly purified MG(T152S), 0.2 mg/ml; 2, MG(T152S) 20 min at 37 °C; 3, MG(T152S) 40 min at 37 °C in reaction buffer containing 1 mM; 4, 0.5 mM; 5, 0.25 mM; 6, 0.125 mM; 7, 62.5 µM; 8, 31.3 µM; 9, 15.6 µM; 10, 7.8 µM of glycine.
Figure 5: Hydroxylamine (HA) effect on autoproteolysis of pre-G(T152C). 1, pre-G (T152C) incubated for 1 h at 37 °C; 2, pre-G(T152C) with 250 mM HA for 1 h at 37 °C; 3, pre-G(T152C) incubated with 10 mM iodoacetamide (IAA), overnight at 10 °C; 4, IAA-treated pre-G(T152C) with HA for 1 h at 37 °C; 5, pre-G(T152S) with IAA, overnight at 10 °C (about 50% pre-G(T152S) was already processed during IAA treatment); 6, IAA-treated pre-G(T152S) with HA, 1 h at 37 °C.
The amino acid sequence HDTIG surrounding the processing site
of glycosylasparaginase is conserved from humans to
bacteria(13) . It is known that in the human enzyme only
His-204 in the above sequence is involved in activation(6) . We
replaced the corresponding histidine residue (His-150) in the
MBP-bacterial enzyme fusion with serine by site-directed mutagenesis
and examined its expression in E. coli. The purified
MBP-mutant fusion protein was a single polypeptide with a mass of 75
kDa and did not undergo autoproteolysis, even in the presence of 250
mM hydroxylamine (data not shown). This suggests that His-150
of the bacterial glycosylasparaginase, like His-204 of the human
enzyme, is involved in autoproteolysis, most likely in the formation of
the ester intermediate. Further evidence for the involvement of
histidine in autoproteolysis came from examining the effect of pH on
the autoproteolysis of pre-MG(T152S). Autoproteolysis was maximal
between pH 6.0 and 7.5, while below pH 4.0 or above pH 9.0 it was not
detectable. This pH dependence is consistent with the involvement of a
histidine side chain, which generally has a pK about 6. It should be noted that this behavior is very different
from the pH-dependent profile for glycosylasparaginase activity, which
extends far into the alkaline range. Glycosylasparaginase activity is
optimal at approximately pH 9.0, where autoproteolysis can barely be
detected. These results suggest that His-150 does not play the same
role in enzymatic activity as in autoproteolysis.
Figure 6:
Measurement of activation energy for
autoproteolysis by Arrhenius plots. The rate constant k of
autoproteolysis of precursor at different temperatures was measured by
SDS-PAGE and gel scanning. The data were plotted as ln k against 1000/T, where T is the absolute
temperature. , pre-MG(T152S);
,
pre-MG.
Non-protease zymogens are generally considered to be processed by proteases. But in this study we have demonstrated that the precursor of glycosylasparaginase is processed by a unique intramolecular autoproteolysis.
This study has identified His-150 and the hydroxyl or thiol group of residue 152 as being involved in autoproteolysis. The optimum pH range for autoproteolysis suggests that His-150 is the proton acceptor-donor base in the reaction. A thioester intermediate is likely formed by Cys-152 of pre-G(T152C) during autoproteolysis. This would indicate that the mechanism of autoproteolysis resembles proteolysis by serine or cysteine proteases. Since glycosylasparaginase does not possess protease activity per se, the activation cannot be an autocatalytic process. It is a spontaneous intramolecular reaction with the key mechanistic characteristics of serine/cysteine proteases.
Based on the data
presented here and in other published studies(5, 6) ,
we propose the following model of autoproteolysis of
glycosylasparaginase, illustrated in Fig. 7. When the newly
synthesized polypeptide is secreted into the periplasm, the signal
peptide is removed and the precursor is properly folded. The reactive
hydroxyl of Thr-152 is deprotonated by His-150 (possibly mediated by a
water molecule), and then launches a nucleophilic attack in cis on the -carbonyl carbon of Asp-151 to form a transitional
tetrahedral intermediate associated with a five-member ring structure.
After a proton is transferred to the leaving amino group of Thr-152,
the
-carbonyl of Asp-151 is shifted to the hydroxyl of Thr-152
leading to the ester intermediate via an N-O shift (20) . The
final step is hydrolysis of the ester by water. During this maturation
process, we assume that residues close to the cleavage site or other
places are reoriented to give the final structure of the active site
and the correct conformation of the mature enzyme.
Figure 7: Model for autoproteolysis of glycosylasparaginase (see text).
Even though the
above model is consistent with the experimental results so far
obtained, many questions remain to be answered. What is the biological
significance of the apparent amino acid binding site of the precursor
associated with inhibition of autoproteolysis? Residue 152 at the
processing site is the active residue for both autoproteolysis and
enzyme activity. Changing Thr-152 to Ser dramatically increases the
inhibition of autoproteolysis by certain amino acids such as glycine.
The mature enzyme is also inhibited by certain amino acids such as
aspartic acid and the asparagine analog
5-diazo-4-oxo-L-norvaline (DONV). The K of glycine for autoproteolysis of pre-MG(T152S), the K
of the natural substrate Asn-GlcNAc, and the K
of DONV for glycosylasparaginase activity are
similar (30-100 µM)(8) . Thus, the simplest
explanation is that the amino acid binding site of the precursor is the
partly formed substrate binding site in the mature enzyme, which
implies that the processing site and the enzyme active center are
located in the same area of the protein.
Why is the active threonine
residue conserved from humans to bacteria? Activation of the active
mutants, but not the wild type enzyme, is inhibited in vivo.
One explanation is that the methyl group of Thr-152 interacts with
another part of the protein and prevents inhibitors such as amino acids
from accessing and binding to the partly formed substrate site and thus
interfering with autoproteolysis. By the same token we can also
interpret the data from the activation energies of autoproteolysis of
the wild type Thr-152 and the T152S mutant. The methyl group of
Thr-152, through an as-yet-unidentified interaction, could prevent free
rotation about the C-C
bond of Thr-152 and place the reactive
hydroxyl in a proper orientation for deprotonation and subsequent
nucleophilic attack, thus reducing the activation entropy. The
activation enthalpies for shifting the
-carbonyl of Asp-151 to the
primary hydroxyl of Ser-152 or to the secondary hydroxyl group of
Thr-152 should be similar. These potential multiple functions of the
methyl group of Thr-152 might explain why this active threonine is
evolutionarily conserved among different glycoasparaginases, and why
the native enzyme is more active than the serine mutant.
In human
glycosylasparaginase, no histidine residues are involved in enzyme
activity(6) . The pH dependence of glycosylasparaginase
activity is not consistent with histidine as a proton acceptor-donor.
Therefore, it is very likely that glycosylasparaginase, like the
-subunits of proteasomes or the E. coli penicillin
acylase, is also a single-amino acid catalytic center enzyme and the
newly generated
-amino group of the N-terminal Thr of
-subunit becomes the proton acceptor-donor for enzyme
activity(21, 22) . Another intriguing question is what
are the actual conformational changes in the protein resulting from
activation that convert the the protein to glycosylasparaginase. X-ray
structural analysis on both the properly folded precursor and the
mature enzyme will be essential to answer these questions.
The mutations of human AGUs so far found which result in a deficiency of activation are not located at the processing site. This suggests that the proper folding and the correct conformation of the precursor play an indispensable role in activation.
Autoproteolytic processes play
important roles in post-translational processing of gene products. For
example, the Hedge-hog proteins undergo a specific autoproteolysis to
realize their biological function (23, 24) . The
precursors of the catalytic -subunits of proteasomes may be
activated by autoproteolysis after being assembled with the
-subunits into the proteasomes(25) . Autoproteolytic
cleavage also serves as a mechanistic component for protein
splicing(20) . Understanding the mechanism of this unique
post-translational process in glycosylasparaginase may not only provide
valuable information for studies related to AGU, it may also shed light
on autoproteolytic activations of different gene products in different
biological systems.