From the Protein Engineering Network of Centres of Excellence of
Canada and the
Department of Chemistry, University of
British Columbia, Vancouver, British Columbia V6T 1Z1, Canada
Mannosidases play a key role in the processing of
glycoproteins and thus are of considerable pharmaceutical interest and
indeed have emerged as targets for the development of anti-cancer
therapies. Access to useful quantities of the mammalian enzymes has not
yet been achieved; therefore, jack bean mannosidase, a readily
available enzyme, has become the model system. However, the relevance
of this enzyme has not been demonstrated, nor is anything known about the active site structure of this, or any other, mannosidase. Hydrolysis by this enzyme occurs with net retention of sugar anomeric configuration; thus, a double displacement mechanism involving a
mannosyl-enzyme intermediate is presumably involved. Two new mechanism-based inhibitors, 5-fluoro-
-D-mannosyl
fluoride and 5-fluoro-
-L-gulosyl fluoride, which
function by the steady state trapping of such an intermediate, have
been synthesized and tested. Both show high affinity for jack bean
-mannosidase (Ki
= 71 and 86 µM,
respectively), and the latter has been used to label the active site
nucleophile. The labeled peptide present in a peptic digest of this
trapped glycosyl-enzyme intermediate was identified by neutral loss
scans on an electrospray ionization triple quadrupole mass
spectrometer. Comparative liquid chromatographic/mass spectrometric
analysis of peptic digests of labeled and unlabeled enzyme samples
confirmed the unique presence of this peptide of m/z 1180.5 in the labeled sample. The label was cleaved from the peptide by
treatment with ammonia, and the resultant unlabeled peptide was
purified and sequenced by Edman degradation. The peptide identified
contained only one candidate for the catalytic nucleophile, an aspartic
acid. This residue was contained within the sequence Gly-Trp-Gln-Ile-Asp-Pro-Phe-Gly-His-Ser, which showed excellent sequence similarity with regions in mammalian lysosomal and Golgi
-mannosidase sequences. These mammalian
-mannosidases belong to
family 38 (or class II
-mannosidases) in which the Asp in the above
sequence is totally conserved. This finding therefore assigns jack bean
-mannosidase to family 38, validating it as a model for other
pharmaceutically interesting enzymes and thereby identifying the
catalytic nucleophile within this family.
 |
INTRODUCTION |
There has been widespread interest in mannosidases in recent
years, largely due to their role in a multitude of biological systems
and, as a result, their potential as therapeutic targets. In
particular, mammalian Golgi
-mannosidase II is involved in glycoprotein biosynthesis and is currently an important therapeutic target for the development of anti-cancer agents (1). Mammalian lysosomal
-mannosidase has significant sequence similarity to the
Golgi class II enzyme and is responsible for glycoprotein degradation
(2, 3). The absence of this enzyme causes the genetic lysosomal storage
disease
-mannosidosis in humans and cattle (4-6). These
mannosidases have been categorized as class II mannosidases, based on
sequence alignment, and clearly belong to family 38 in Henrissat's
glycosidase classification (2, 7-9). Class I
(1,2)-mannosidases
have little similarity in sequence or substrate/inhibitor specificity
to the class II enzymes and are classified as family 47 (2, 7, 10).
While a number of these enzymes have been cloned and sequenced,
relatively little success has been attained in their high level
expression. This has severely limited structural and mechanistic
studies on this important enzyme class, significantly slowing the
search for potential therapeutic agents based upon their inhibition. In
their absence, a suitable model enzyme is necessary, and the jack bean
enzyme has been assumed to suit that role, although this choice has not been validated.
Jack bean
-mannosidase, although a commercially available enzyme,
has not been characterized structurally, nor has its primary sequence
been determined. Like the lysosomal enzyme, it hydrolyzes
(1,2),
(1,3), and
(1,6) linkages between mannose residues but also has a
broad aglycone specificity (19). In common with class II
-mannosidases, the jack bean enzyme is a retaining enzyme (14),
releasing
-D-mannose as first formed product. As such, it is presumed to follow a double-displacement mechanism in which a
glycosyl-enzyme intermediate is formed and hydrolyzed via oxocarbenium ion-like transition states (11, 12, 15, 16). Formation of this
intermediate is assisted by general acid catalysis from a carboxylic
acid located in the active site. This same residue serves as the
general base catalyst for the second, deglycosylation, step. A second
active site carboxylic acid serves as the nucleophile that forms the
covalent intermediate. The jack bean enzyme, like other class II
mannosidases, is inhibited by swainsonine and mannostatin (17, 18) and
accepts aryl-mannoside substrates. In contrast, the class I
(1,
2)-mannosidases are inverting enzymes and possess none of these other
characteristics (10, 13). Since only two families of
-mannosidases
have been established to date, families 38 and 47, of which the former
is retaining, it is quite possible that the jack bean enzyme, being a
retaining enzyme, may well also be a member of family 38. However,
sequence information will be necessary to confirm this prediction.
The nucleophilic and acid/base active site residues have not been
labeled or identified in any
-mannosidase, nor has any crystal
structure been obtained. Jack bean
-mannosidase was therefore chosen
for use in the development of new methodology aimed at labeling and
identifying catalytically important residues in this enzyme class.
2-Deoxy-2-fluoro-
-D-glycosyl fluorides have proved to be
valuable reagents for identifying the active site nucleophiles in a
range of retaining
-glycosidases (20-22). However, the labeling of
retaining
-glycosidases with these compounds has been unsuccessful, leading to the development of 5-fluoroglycosyl fluorides to solve this
problem (23, 24). These fluorosugars behave as mechanism-based inactivators. The good fluoride leaving group at C-1 serves to accelerate the first step of the double displacement reaction, whereas
the C-5 or C-2 fluorine slows both steps via inductive destabilization
of the carbocationic transition states and results in the trapping of a
5-fluoro- or 2-deoxy-2-fluoroglycosyl-enzyme intermediate.
5-Fluoro-
-D-glucosyl fluoride and
5-fluoro-
-L-idosyl fluoride have been used to label and
identify Asp-214 as the active site nucleophile in
-glucosidase from
Saccharomyces cerevisiae (24). Here we describe the
application of a new label, 5-fluoro-
-L-gulosyl fluoride
(5FguloF)1 to label the
active site nucleophile in jack bean
-mannosidase. The
identification of the catalytic nucleophile is then made possible by
proteolytic digestion of the labeled enzyme followed by HPLC separation
of the resultant peptides and localization of the labeled peptide using
electrospray ionization tandem mass spectrometry to detect specific
fragmentations associated with the glycosylated active site peptide
(22, 25).
 |
EXPERIMENTAL PROCEDURES |
General Procedures and Synthesis--
Jack bean
-mannosidase,
all buffer chemicals, and other reagents were obtained from Sigma
unless otherwise noted. Pepsin (from porcine mucosa) was obtained from
Boehringer Mannheim.
2,4-Dinitrophenyl-
-D-mannopyranoside (DNPM) was
synthesized in one step by treatment of D-mannose with 1-fluoro-2,4-dinitrobenzene (26). Synthesis of the inhibitors was
performed according to Scheme 1, as
follows.
2,3,4,6-Tetra-O-acetyl-5-bromo-
-D-mannopyranosyl
fluoride
(4)--
2,3,4,6-Tetra-O-acetyl-
-D-mannopyranosyl
fluoride (3) (15.3 g) and N-bromosuccinimide (31 g, 17.4 mmol) were suspended in carbon tetrachloride and heated at
reflux under nitrogen by means of two 200-watt household light bulbs.
After 5 h, the mixture was allowed to cool and was filtered. The
filtrate was washed with saturated sodium hydrogen carbonate (400 ml)
and brine (400 ml), dried (MgSO4), and then filtered. The
solvent was removed in vacuo, and the residue was purified
on silica gel (dichloromethane, 3% acetonitrile) to give
2,3,4,6-tetra-O-acetyl-5-bromo-
-D-mannopyranosyl fluoride (10 g, 23.3 mmol, 53%) as a colorless syrup. 1H
NMR (400 MHz, CDCl3)
5.67 (1 H, dd,
J2,3 = 3.2 Hz, J3,4 = 10.1 Hz, H-3) 5.65 (1 H, dd, J1,F1 = 48.5 Hz,
J1,2 = 1.7 Hz, H-1), 5.48-5.54 (2 H, m, H-2 and
H-4), 4.40 (2 H, s, H-6 and H-6
), 2.4 (3 H, Me), 2.09 (6 H, 2 × Me), 1.99 (3 H, s, Me). CIMS (NH3): m/z 446 (M + NH4)+ (100%), 448 (M + NH4)+ (100%)
2,3,4,6-Tetra-O-acetyl-5-fluoro-
-L-gulopyranosyl
Fluoride
(5)--
2,3,4,6-Tetra-O-acetyl-5-bromo-
-D-mannopyranosyl
fluoride (4) (1.5 g, 3.5 mmol) was dissolved in
acetonitrile (20 ml) and stirred with silver fluoride (890 mg, 7.1 mmol) in darkness under nitrogen. After 16 h, the mixture was
filtered through a silica/Celite plug, which was then washed with ethyl
acetate (100 ml). The organic layer was washed successively with
saturated sodium hydrogen carbonate (60 ml), water (60 ml), and brine
(60 ml). The organic layer was dried (MgSO4) and filtered,
and the solvent was removed in vacuo. Purification on silica
gel (ethyl acetate:petroleum ether; b.p. 35-60 °C; 1:4) gave
2,3,4,6-tetra-O-acetyl-5-fluoro-
-L-gulopyranosyl fluoride (712 mg, 1.93 mmol, 55%) and starting material (214 mg, 5.0 mmol) as a colorless syrup. [
]D +37.5° (c
1.5, CHCl3); 19F NMR (188 MHz,
CDCl3, CF3CO2H reference)
:
75.37 (dd, JF1,H1 = 53.2 Hz,
JF1,H2 = 10.5 Hz, F-1),
42.15 (ddd,
JF5,H4 = 4.4 Hz, JF5,H6
= 23.6 Hz, JF5,H6
= 13.8 Hz, F-5);
1H NMR (400 MHz, CDCl3)
5.85 (1 H, dd,
J1,2 = 7.3 Hz, H-1), 5.38 (1 H, dd,
J3,2 = 3.6 Hz, J3,4 = 4 Hz, H-3), 5.32 (1 H, dd, H-4), 5.22 (1 H, ddd, H-2), 4.49 (1 H, dd,
J6,6
12.3 Hz, H-6), 4.15 (1 H, dd, H-6
); CIMS
(NH3): m/z 386 (M + NH4)+ (100%),
349 (M
F)+ (20%). Anal. Calcd for
C14H18O9F2: C, 45.65; H, 4.92. Found: C, 45.90; H, 5.03.
2,3,4,6-Tetra-O-acetyl-5-fluoro-
-D-mannopyranosyl
Fluoride (6)--
Boron trifluoride diethyletherate (1 eq)
was added to a stirred solution of
2,3,4,6-tetra-O-acetyl-5-fluoro-
-L-gulopyranosyl fluoride (220 mg, 0.60 mmol) in dichloromethane (8 ml). After 4 h,
saturated sodium hydrogen carbonate (10 ml) and dichloromethane (10 ml)
were added. The organic layer was further washed with brine (10 ml) and
dried (MgSO4), filtered, and the solvent was removed
in vacuo. The residue was purified on silica gel (ethyl acetate:petroleum ether; b.p. 35-60 °C; 1:3) to give the product 2,3,4,6-tetra-O-acetyl-5-fluoro-
-D-mannopyranosyl
fluoride (119 mg, 54%) as needles. m.p. 79-80 °C
(dichloromethane/diethylether/petroleum ether; b.p. 35-60 °C).
[
]D
23.3° (c 0.9, CHCl3);
19F NMR (188 MHz, C6D6,
CF3CO2H reference)
51.12 (ddd,
JF1,H1 = 48.5 Hz, JF1,H2 = 2.4 Hz, JF1,F5 = 22.8 Hz, F-1),
47.97 (dddd, JF5,H4 = 22 Hz, JF5,H6 = 7.8 Hz, JF5,H6
= 4.3 Hz, F-5); 1H
NMR (400 MHz, C6D6)
5.97 (1 H, dd,
J3,2 = 2.8 Hz, J3,4 = 10.8 Hz, H-3), 5.86 (1 H, dd, H-4), 5.64 (1 H, dt,
J2,1 = 2.4 Hz, H-2), 5.12 (1 H, dd, H-1), 4.49 (1 H, dd, J6,6
= 12.1 Hz, H-6), 4.07 (1 H, dd,
H-6
); CIMS (NH3): m/z 386 (M + NH4)+(100%), 349 (M
F)+
(80%). Anal. Calcd for
C14H18O9F2: C, 45.65;
H, 4.92. Found: C, 45.51; H, 5.02.
5FguloF
(2)--
2,3,4,6-Tetra-O-acetyl-5-fluoro-
-L-gulopyranosyl
fluoride (340 mg, 0.92 mmol) was dissolved in methanol (15 ml) and
cooled to
5 °C. Ammonia gas was bubbled through the solution for 5 min, after which the flask was sealed and allowed to warm to room
temperature After 2 h, the solvent was removed in
vacuo, and the residue was purified on silica gel (ethyl
acetate:methanol:water; 27:2:1) to give 5FguloF (152 mg, 0.76 mmol,
82%) as a colorless syrup. Anal. Calcd for
C6H10O5F2: C, 36.01; H,
5.04. Found: C, 36.26; H, 5.11. 19F NMR (188 MHz,
D2O, CF3CO2H reference)
68.29
(d, JF1,H1 = 52.4 Hz, F-1),
43.66 (m, F-5).
1H NMR (400 MHz, D2O, DSS reference)
5.72 (1 H, dd, J1,2 = 5.1 Hz, H-1), 4.06-4.10 (2 H,
m, H-3, H-4), 4.00 (1 H, m, H-2), 3.93 (1 H, dd,
J6,6
= 13.1 Hz, J6,F5 = 15.5 Hz, H-6), 3.73 (1 H, dd, J6
,F5 = 25.2 Hz,
H-6
). Anal. Calcd for
C6H10O5F2: C, 36.01; H,
5.04. Found: C, 36.26; H, 5.11.
5-Fluoro-
-D-mannopyranosyl Fluoride (5FmanF)
(1)--
This was prepared from
2,3,4,6-tetra-O-acetyl-5-fluoro-
-D-mannopyranosyl
fluoride (117 mg, 0.109 mmol) in an analogous fashion to 5FguloF to
give 5FmanF (59 mg, 0.041 mmol, 93%) as a colorless syrup.
veLSIMS:
199 (M
H). (M
H): Calcd, 199.0415; found, 199.04215. 19F NMR (188 MHz, D2O,
CF3CO2H reference)
52.49 (dd,
JF1,H1 = 49.3 Hz, JF1,F5 = 22 Hz, F-1),
54.00 (tt, JF5,H4 = 22 Hz,
JF5,6 = 10 Hz, JF5,6
= 10 Hz, F-5). 1H NMR (400 MHz, D2O, DSS
reference)
5.72 (1 H, d, H-1), 4.24 (1 H, m, H-2), 4.13 (1 H, dd,
J3,2 = 1.8 Hz, J3,4 = 10.3 Hz, H-3), 4.01 (1 H, dd, H-4), 3.81 (2 H, d, H-6 and H-6
).
veLSIMS: (M
H): Calcd, 199.0415; found, 199.0421.
Enzyme Kinetics--
Kinetic studies were performed at 25 °C
except for the inactivation experiment with 5FguloF, which was
performed at 4 °C. All studies were performed in 50 mM
sodium citrate buffer, pH 4.5, containing 0.1% bovine serum albumin. A
continuous spectrophotometric assay based on the hydrolysis of DNPM was
used to monitor enzyme activity by measurement of the rate of
2,4-dinitrophenolate release (
= 400 nm:
= 8439 ± 261 M
1 cm
1 in the buffer above)
using a UNICAM 8700 UV-visible spectrophotometer equipped with a
circulating water bath.
The inactivation of
-mannosidase by 5FguloF was monitored by
incubation of the enzyme (~0.02 mg/ml) under the above conditions in
the presence of various concentrations of 5FguloF at 4 °C. Residual
enzyme activity was determined at the appropriate time intervals by
addition of a 10-µl aliquot of the inactivation mixture to a solution
of DNPM (0.5 mM, 750 µl) in the above buffer and measurement of dinitrophenolate release over a period of 30 s. Pseudo-first-order inactivation rate constants at each 5FguloF concentration (kobs) were determined by fitting
the initial exponential phase of each curve to a first-order equation
using the program GraFit (Leatherbarrow, R. J. GraFit
version 3.0; Erithacus Software Ltd.: Staines, United Kingdom, 1990).
The value of ki/KI, assuming
inactivation according to the kinetic model shown in Scheme
2, was determined from the slope of a
plot of kobs against time.
The kcat value for 5FguloF was determined by
inactivation of the enzyme (0.01 mg/ml) in the presence of 0.8 mM 5FguloF followed by the addition of a 10-µl aliquot of
the enzyme to a solution of DNPM (800 µl). The release of
dinitrophenolate was measured continually over consecutive 15-s periods
up to ~7 min. The average rate of dinitrophenolate release was
calculated for the 15 s periods and plotted as a function of time,
thereby providing a measure of the rate of return of activity due to
turnover. A value for the first-order rate constant for reactivation
(kcat) was determined by fitting the curve to
Equation 1. This was repeated at various concentrations of DNPM. In
addition, the kcat values for 5FguloF and 5FmanF
were determined directly by monitoring the release of fluoride using an
Orion 96-09 combination fluoride electrode.
|
(Eq. 1)
|
The apparent dissociation constants
(KI
) for the interaction of
5FguloF and 5FmanF with the enzyme under steady-state reaction
conditions were determined by continuous measurement of
dinitrophenolate release in the presence of DNPM and inhibitor. This
was repeated at several different concentrations of inhibitor, the
enzyme first being allowed to react for 5 min before the assay was run
to ensure that a steady state was achieved. The observed rates were
plotted in the form of a Dixon plot (1/v versus
[I]), and the KI
value was
determined from the intercept of this line with the horizontal line
drawn through 1/Vmax.
Labeling and Proteolysis--
Labeling of jack bean
-mannosidase was achieved by incubating the enzyme (1 mg/ml × 20 µl) with 5FguloF (1 µl × 40 mM) for 10 min in
50 mM citrate buffer (pH 4.5). This sample was then used
directly for mass spectrometric analysis. When prepared for proteolytic
digestion purposes, the inactivation was repeated using a more
concentrated enzyme sample (5 mg/ml × 20 µl). After incubating
for 10 min, the sample was diluted with pepsin solution (60 µl × 0.1 mg/ml pepsin; 50 mM sodium phosphate/HCl, pH 1.8), and the mixture was incubated at room temperature for 30 min. ESMS
analysis and SDS-polyacrylamide gel electrophoresis of the proteolytic
digest confirmed that the enzyme was completely digested.
Electrospray Mass Spectrometry--
The analyses of the protein
and peptide samples were carried out using a Sciex API-300 mass
spectrometer interfaced with a Michrom UMA HPLC system (Michrom
Bioresources, Inc., Auburn, CA). Intact jack bean
-mannosidase
(10-20 µg, labeled or unlabeled) was introduced into the mass
spectrometer through a microbore PLRP column (1 × 50 mm) and
eluted with a gradient of 20-100% solvent B at a flow rate of 50 µl/min over 5 min (solvent A, 0.06% trifluoroacetic acid, 2%
acetonitrile in water; solvent B, 0.05% trifluoroacetic acid, 90%
acetonitrile in water). The MS was scanned over a range of 400-2300 Da
with a step size of 0.5 Da and a dwell time of 1 ms.
The peptides were analyzed by loading a 10-µl sample of the pepsin
digest (1.25 mg/ml) onto a C18 column (Reliasil, 1 × 150 mm),
which was eluted at a flow rate of 50 µl/min with a gradient of
0-60% B over 45 min. The proteolytic mixture was first examined in
LC/MS mode and then in the neutral loss mode.
In the single quadrupole (normal LC/MS) mode, the MS conditions were as
follows. The mass analyzer was scanned over the range of 300-2400 Da
with a step size of 0.5 Da, a dwell time of 1.5 ms, an ion source
voltage of 4.8 kV, and an orifice energy of 50 V. The neutral loss
spectra were obtained in the triple quadrupole mode searching for the
loss of m/z 60.3, which corresponds to the loss of the
inhibitor label from a peptide that is triply charged. A scan range of
400-1800 Da was used with a step size of 0.5 Da and a dwell time of
1.5 ms. Other parameters were as follows: ion source voltage = 5.5 kV, orifice energy = 45 V, IQ2 =
50, Q0 =
10,
CAD = 4.
Chemical Sequencing--
Partial purification of the labeled
peptide was achieved by HPLC separation of the pepsin digest as
described above and by collecting the appropriate fractions containing
the partially purified labeled peptide via a postcolumn splitter. The
label was cleaved by treatment with aqueous ammonia (100 µl of
sample/10 µl of concentrated aqueous ammonia/10 min), and the
resulting unlabeled peptide was purified by HPLC using the same
conditions as above. Loss of the label increases the retention time by
~40 s, which allows isolation of pure material The amino acid
sequence of the labeled peptide was determined by S. Perry of the
Nucleic Acid and Peptide Service at the University of British Columbia using standard pulsed liquid phase protocols and instrumentation on a
Perkin-Elmer model 476A sequencer and model 120A phenylthiohydantoin analyzer (Applied Biosystems, Foster City, CA).
Methyl Esterification of Partially Purified Unlabeled
Peptide--
Partially purified unlabeled peptide (control sample) was
mixed with a freshly prepared solution of 2 M methanolic
HCl, and the mixture was incubated at room temperature for 30 min. The excess reagent was removed by concentration under vacuum (SVC 100 Speed
Vac), and the product was dissolved in 50% acetonitrile/water.
 |
RESULTS AND DISCUSSION |
Synthesis--
5FmanF (1) and 5FguloF (2)
(Scheme 1 and Fig. 1) were synthesized
from 2,3,4,6-tetra-O-acetyl-
-D-mannosyl
fluoride (3) by four-step and three-step procedures as
follows (Scheme 1). Radical bromination of
2,3,4,6-tetra-O-acetyl-
-D-mannosyl fluoride
(3) in carbon tetrachloride generated the 5-bromo derivative
(4). Displacement of the bromine at C-5 using silver
fluoride in acetonitrile yielded the product of inverted configuration
at C-5, namely
2,3,4,6-tetra-O-acetyl-5-fluoro-
-L-gulopyranosyl fluoride (5) in good yield. Subsequent treatment of this compound with borontrifluoride diethyl etherate in dichloromethane resulted in equilibration of the C-5 stereochemistry, with production of the more stable
2,3,4,6-tetra-O-acetyl-5-fluoro-
-D-mannopyranosyl fluoride (6). Deprotection of both 5 and
6 was achieved by treatment with ammonia in methanol to
yield, after purification, analytically pure materials.
1H NMR analysis reveals that 5FguloF (2), the
C-5 epimer of the mannocompound (1), adopts a
"boat-like" conformation, whereas 5FmanF adopts a normal
4C1 chair conformation. This is supported by an
x-ray crystal structure of the tetraacetate derivative of 1 (not shown), which, again, clearly shows a normal chair
conformation.
Kinetic Studies--
5FGuloF (2) inactivates jack bean
-mannosidase in a time-dependent fashion as shown in
Fig. 2a, with inactivation at
higher concentrations occurring too fast to measure, although the
experiment was conducted at 4 °C (Fig. 2a). Inactivation
is assumed to follow the model shown in Scheme 2. Pseudo-first-order rate constants were determined for inactivation at each concentration of inhibitor from the slopes of these plots, and a replot of these rate
constants versus inhibitor concentration was found to be linear (Fig. 2b), allowing a second order rate constant,
ki/Ki, of 0.017 s
1
mM
1 to be obtained from the slope of this
plot. The absence of any saturation behavior in this plot indicates a
Ki value in excess of 0.3 mM.
Unfortunately, the rapid rate of inactivation precluded determination
of either this parameter or the maximal inactivation rate constant,
ki. However, evidence for active site binding was
obtained from the protection against inactivation afforded by 0.18 mM mannojiritetrazole, a known competitive inhibitor of
-mannosidases (Ki = 0.18 mM) (27). In
its presence, the pseudo-first-order rate constant for inactivation at
0.19 mM 5FguloF was reduced from 1.9 × 10
3 s
1 to 0.75 × 10
3
s
1.

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Fig. 2.
Inactivation of jack bean -mannosidase by
5FguloF (2). a, semilogarithmic plot of residual
activity versus time at the following inactivator
concentrations: 0.05 ( ), 0.1 ( ), 0.15 ( ), 0.2 ( ), and 0.3 mM ( ). V, rate of DNPM hydrolysis.
b, plot of pseudo-first-order rate constants from a
versus [5FguloF].
|
|
These data therefore indicate that, as seen in previous studies with
2-fluoroglycosyl fluorides on
-glycosidases (20-22), the 5-FguloF
is inactivating the enzyme by trapping of a fluoroglycosyl-enzyme intermediate. Further insight was obtained by demonstrating the catalytic competence of the intermediate so trapped through the time-dependent reactivation observed when the labeled
enzyme was freed from excess inhibitor (Fig.
3). Enzyme in the presence of 0.8 mM 5FguloF was diluted 80-fold into substrate (DNPM), and the reactivation was monitored continually over time. Such reactivation could be occurring via hydrolysis of the 5-fluorogulosyl-enzyme intermediate or via transglycosylation to DNPM. However, as can be seen
in Fig. 3, the rate of reactivation was independent of substrate (DNPM)
concentration. This suggests that DNPM does not bind significantly to
the glycosyl-enzyme, thus that reactivation proceeds via a hydrolytic
process. A first order rate constant for reactivation at 25 °C,
kcat, of 9.6 × 10
3
s
1 was calculated.

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Fig. 3.
Reactivation of inactive jack bean
-mannosidase in the presence of DNPM. a, rate of change
of absorbance at 400 nm ( enzyme activity) versus time in
the presence of DNPM at concentrations of 1 ( ), 2 ( ), 4 ( ), 8 ( ), 12 ( ), and 16 mM ( ). b, first-order reactivation rate constants from a versus [DNPM].
|
|
Confirmation of the value of this rate constant for the hydrolytic
process was obtained by another method. Since turnover is observed,
5FguloF must be acting as a substrate, albeit a slow one. It was
therefore possible to monitor substrate activity directly by monitoring
fluoride release using a fluoride-selective electrode. The
5-fluorogulose released rapidly decomposes with release of a second
equivalent of fluoride; thus, all steady state kinetic data have been
calculated on the basis of 2 eq of fluoride being released per
substrate molecule hydrolyzed. Using this approach, the steady-state
rate of release of fluoride was determined, and a value of 7.9 × 10
3 s
1 was calculated for
kcat. This is very close to the value determined from reactivation kinetics, showing that both methods are monitoring the same hydrolytic process. These data are therefore consistent with a
model in which 5FguloF forms a short lived (t1/2 = 72 s) 5-fluorogulosyl-enzyme intermediate that accumulates in a
time-dependent fashion, since the rate of formation
(governed by ki) is much greater than the rate of
hydrolysis (kcat).
Since 5FguloF forms a significant steady state concentration of
glycosyl-enzyme intermediate, thereby blocking the active site, it can
also be studied as a competitive inhibitor. The apparent dissociation
constant, KI
(Equation 2) can
therefore be determined by measuring the rate of hydrolysis of DNPM in
the presence of various concentrations of 5FguloF. The value of
KI
= 86 ± 14 µM determined for 5FguloF represents a minimum value for
the true dissociation constant KI, as defined below (28).
|
(Eq. 2)
|
Somewhat different kinetic behavior was seen with 5FmanF
(1), which did not show time-dependent
inactivation behavior but rather was found to reversibly inhibit jack
bean
-mannosidase with an apparent
KI
of 71 ± 13 µM. This behavior suggests that 5FmanF (1) is
also acting as a slow substrate but that this time the turnover is too
fast to allow trapping of the intermediate (therefore inactivation) on
the time scale of the assay employed. Indeed, the enzyme was shown to
slowly hydrolyze (1), the slow release of fluoride being
measured with a fluoride-selective electrode. The rate of fluoride
release was found to be independent of the concentration of 5FmanF at
concentrations down to 0.7 mM, with a value of 0.025 ±0.002 s
1 being measured for
kcat. The apparent tight binding of 1 is thus once again a consequence of the accumulation of an
intermediate.
Identification of the Site of Attachment of the
Label--
Labeling studies with jack bean
-mannosidase to identify
the catalytic nucleophile were carried out with 5FguloF, since it forms
a longer lived glycosyl-enzyme intermediate than does 5FmanF. The
stoichiometry of inactivation by 5FguloF was determined by application
of electrospray mass spectrometry. Although native jack bean
-mannosidase has a molecular weight of 230,000 (29), SDS-polyacrylamide gel electrophoresis indicated two major subunits, one of molecular weight 66,000 (comparison with bovine serum albumin) and one of lower molecular weight around 40,000-50,000. Mass
spectrometric analysis of native enzyme showed peaks in the 44,000 region and the 66,000 region (Fig.
4a). MS analysis of the
inactive enzyme in the presence of 1 mM 5FguloF showed
peaks at 43,846, 43,959, 66,533, and 66,708 (Fig. 4b). The
increase in molecular weight of the larger subunit (average mass
increase = 171) corresponds well, within experimental error, to
the mass increase of 181 expected upon derivatization with a single
molecule of 2 per active site. This suggests that
catalytically competent active sites occur only in the larger subunit
of this enzyme. The error in mass determination using this method is at
least ±10 Da. This is primarily due to the broad, and somewhat
irregular, profile of the peaks as determined by computer
reconstruction of the initial mass spectrum. Reasons for the
"doublet" nature of the peaks could include variation in
glycosylation or other post-translational modification of the
protein.

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Fig. 4.
Electrospray mass spectrometry of jack bean
-mannosidase. a, reconstructed mass spectrum of unlabeled
enzyme; b, reconstructed mass spectrum of 5FguloF-labeled
enzyme.
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Peptic digestion of the labeled enzyme gave a complex mixture of
peptides that was separated by HPLC using the ESMS as a detector in the
LC/MS mode (Fig. 5a). The
labeled peptide was identified by repeating this process using tandem
mass spectrometry in neutral loss mode. In this mode, ions were
subjected to limited fragmentation by collision with an inert gas,
which caused selective homolytic fission of the labile glycosidic
linkage between the label and the peptide. As a result, the labeled
peptide loses a neutral sugar. The two quadrupoles were scanned in a
linked mode so that only those ions differing by the mass of the label
could be detected. For a singly charged peptide, this m/z
difference is the mass of the label (m/z 181); for the
triply charged peptide, the m/z difference is one-third of
the mass of the label (m/z 60.3). Two charged fragments were
seen to undergo a neutral loss of 60.3 (Fig. 5b) when a
sample of the peptic digest of the labeled enzyme was subjected to this
analysis. However, one of these fragmentations was observed in a
control digest of unlabeled enzyme (Fig. 5c). This left only
one charged fragment (m/z 787.5), which was not present in a
control experiment (Fig. 5d). This corresponded to a labeled
peptide fragment of mass 2359, which elutes with a retention time of
25.2 min.

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Fig. 5.
LC/MS experiments on jack bean
-mannosidase peptic digest. a, labeled with 5FguloF, in
the normal MS mode; b, labeled with 5FguloF, in the neutral
loss mode; c, unlabeled, in the neutral loss mode.
d, mass spectrum of peptide at 25.2 min in panel
b.
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The relevance of this neutral loss peak was checked by inspection of
the peptide compositions of the digests of labeled and unlabeled enzyme
in the standard LC/MS mode. Indeed, a peak corresponding to the labeled
peptide (m/z 1180.5, doubly charged) was seen in the labeled
digest that was not present in that from an unlabeled sample.
Similarly, a strong peak of m/z 1090, doubly charged, corresponding to the nonlabeled peptide, was observed in the unlabeled digest and also in the labeled digest, although at much lower intensity.
Unfortunately, it did not prove possible to determine the sequence of
this peptide by further tandem mass spectrometric analysis. It was
therefore necessary to purify the peptide and subject it to Edman
degradation. A sample of the labeled peptide was therefore partially
purified using the conditions used for the profile in Fig.
4a. Using these conditions, it proved impossible to fully purify the peptide. The partially purified sample was therefore treated
with aqueous ammonia to cleave the ester linkage between the sugar and
the peptide, thereby shifting its retention time away from those of the
contaminating peptides. Further HPLC of the mixture gave a pure sample
of the unlabeled peptide, mass = 2178, which was observed as the
doubly and triply charged peptides, m/z 1090 and 727, respectively. The pure, cleaved peptide was subjected to Edman
degradation, yielding the sequence information shown in Table
I. The sequence was determined to be
NKIPRAGWQIDPFGHSAVQG (calculated mass = 2178), which contains 1 aspartic acid, 1 asparagine, and 2 glutamine residues.
It seemed probable that the aspartic acid, being the only carboxylic
acid residue, was a good candidate for the catalytic nucleophile by
analogy with all retaining glycosidases to date (11, 22). However, it
is possible that, upon cleavage of the label with ammonia, the
catalytic nucleophile (either Glu or Asp) in its ester form could be
chemically modified to an amide functionality (Gln or Asn,
respectively), thereby leaving some ambiguity, since the protein
sequence was not known. To test this, the 2178-Da peptide fragment from
the control, unlabeled, enzyme digest was treated with acidic methanol
to esterify the carboxylic acid groups. The m/z of this
doubly charged peptide increased from 1090 to 1104, which corresponds
to a peptide mass increase of 28. This was conclusive evidence that two
carboxylic acid groups were present, the carboxyl terminus and Asp.
This result confirms that no chemical modification occurred due to
cleavage of the labeled peptide with ammonia and that the catalytic
nucleophile is indeed the aspartic acid.
This sequence showed excellent similarity with sequences from class II
-mannosidases, all of which belong to family 38 (Table II). In addition, this particular
sequence contains one of the five aspartate residues that are conserved
throughout the class II Golgi and lysosomal
-mannosidases. In
particular, the GWQIDPFGHS sequence shows very high similarity
throughout these Golgi and lysosomal
-mannosidases, and the DPFG
sequence itself is completely conserved. It is reasonable to conclude
that jack bean
-mannosidase is a class II enzyme and therefore also
a member of family 38. It is also of interest that the active site
nucleophile of yeast
-glucosidase is located within the sequence
IDTAG, similar to that of IDPFG seen here. Possibly this indicates some
similarity in structure between family 38
-mannosidases and family
13
-glucosidases. The latter are known to have an
(
/
)8 barrel structure.
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Table II
Jack bean -mannosidase peptide sequence containing the nucleophilic
catalyst (underlined) compared with the highly conserved region from
four class II (family 38) -mannosidases
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Interesting comparisons with the molecular weights of other class II
enzymes can also be drawn. Human lysosomal
-mannosidase is initially
synthesized as a polypeptide of ~110 kDa that is subsequently
processed into two subunits of 40-46 kDa and 63-67 kDa, which then
constitute the native protein (molecular mass, 210 kDa) (3). The rat
Golgi
-mannosidase II is a dimer composed of 124-kDa subunits (2).
Treatment with chymotrypsin causes limited proteolysis to give a dimer
of 110-kDa subunits that retains full activity. It seems likely that
jack bean
-mannosidase is also synthesized as a polypeptide chain
of ~ 110 kDa, which forms a dimer. The fragments of mass ~44
and ~66 kDa, observed by ESMS and SDS-polyacrylamide gel
electrophoresis, could be due to limited proteolysis of the protein,
which is still able to maintain its integrity unless exposed to
denaturing conditions.
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CONCLUSION |
5FGuloF is an effective time-dependent inactivator of
jack bean
-mannosidase, forming a relatively short lived
(t1/2 = 72 s) 5-fluorogulosyl-enzyme
intermediate. It therefore ultimately functions as a very slow
substrate. 5FManF also acts as a slow substrate with a submillimolar
Km value but with a much faster turnover rate. The
5-fluorogulosyl-enzyme intermediate formed using 5FgulF was
sufficiently stable to allow direct observation of the labeled enzyme
by electrospray mass spectrometry and subsequent identification of the
active site nucleophile by tandem mass spectrometric analysis of
proteolytic digests coupled with Edman degradation. Inspection of the
amino acid sequence surrounding the catalytic nucleophile showed
excellent similarity with a series of class II Golgi and lysosomal
-mannosidases and suggests that the jack bean enzyme is closely
related to this class of
-mannosidases. Further, the identification
of the catalytic nucleophile as the aspartic acid within the sequence
IDPFGH thereby identifies this as a key residue within family 38, a
group of enzymes of considerable pharmaceutical interest. This result
therefore opens the possibility of facile generation of inactive
mutants of these medically important enzymes for use in "knockout"
studies as well as providing information on the active site that is of
value in understanding the mutations resulting in mannosidosis.