From the
Woodward's reagent K (WRK) completely inactivated
Escherichia coli uridine phosphorylase by reversible binding
in the active site ( K
Uridine phosphorylase (UPase)
Conversion of 5-fluorouracil to 5-fluorouridine or to
5-fluorodeoxyuridine by human UPase is essential for its antitumor
activity. An active search is going on for new compounds that can be
used to selectively inhibit the activity of human and murine UPases
during chemotherapy of some solid tumors (Schwartz et al.,
1985; Naguib et al., 1987; Veres et al., 1988; Iigo
et al., 1990; Komissarov et al., 1993).
Nevertheless, little is known about the groups involved in catalysis
and/or substrate and product binding at the UPase active site. Previous
studies are indicative of histidine (Drabikowska and Wozniak, 1990) and
cysteine (Vita et al., 1986; Bose and Yamada, 1974) residues
and an amino group (Komissarov et al., 1994a) near or in the
active site. We have also investigated the effects of ionic strength
and of a bipolar aprotic solvent on the rate of enzyme-catalyzed
uridine phosphorolysis and have selectively modified both uridine- and
phosphate-binding subsites to demonstrate that active site
hydrophobicity increases upon enzyme-substrate complex formation
(Komissarov et al., 1994b). The data obtained support the view
that enzymatic uridine phosphorolysis proceeds by the
S
In this paper, we report the
kinetic mechanism of selective modification of E. coli uridine
phosphorylase with Woodward's reagent K and describe the location
of an important dicarboxylic amino acid residue in the enzyme active
site.
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy where E is UPase, I is WRK, E*I is the complex of
UPase with WRK, E-I is the modified enzyme,
K
To examine the protection mechanism of the product
(Ura) and substrate (P
As is evident from Fig. 1, WRK inactivates the E.
coli UPase with pseudo first-order kinetics. The deviation from
linearity in semilog plots (Fig. 1 A) after 4 min is most
likely due to reagent decomposition. The data in Fig. 1 B comply with the two-step mechanism in , with
K
Analysis of the effect of ligands on the inactivation with
WRK () revealed three subsites (uracil, phosphate, and
ribose) in the enzyme active center, just as expected from the
chemistry of uridine phosphorolysis. The addition of phosphate or
uracil (one subsite filled) or uridine or deoxyribose-1-phosphate (two
subsites filled) alone at concentrations exceeding the respective
K
Comparison of with the relevant
literature data (I) demonstrates that WRK has an unusually
high affinity for the UPase active site, and the inhibition constant is
at least an order of magnitude lower than for any other enzyme tested.
It is noteworthy that upon filling of the uracil subsite the
K
HPLC analysis of the proteolytic digests (Fig. 4) provides
final proof that WRK reacts with a single dicarboxylic amino acid in
the UPase subunit (incidentally, the minor peaks at 340 nm most likely
account for the slight excess over equimolarity seen in
Fig. 2B). As shown by Edman degradation, it is Asp
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy Its selective modification with WRK completely inactivates the
enzyme. However, the functional role of this residue remains uncertain,
because activity could be lost simply because of steric hindrances from
a bulky reagent in the active site.
The activated ester generated by
WRK-carboxyl interaction is readily subject to nucleophilic attack
(Fig. 5). Thus consecutive treatment of UPase with WRK and
hydroxylamine results in removal of the WRK residue and restores the
enzyme specific activity from <1 to 20% of the initial. Being again
exposed to WRK, such enzyme retains 9% of the initial activity (45% of
the 20%) and is not noticeably reactivated with hydroxylamine. Thus,
the enzyme preparation obtained after the first round of
WRK/hydroxylamine treatment appears to be a mixture of 10% native
(restituted; specific activity about 110 units/mg) and 90% hydroxamic
UPase (specific activity about 10 units/mg). Only the native form
reacts with WRK in the second exposure, and subsequent treatment with
hydroxylamine leads to quantitative replacement of the Asp
Comparison of the kinetic parameters for the native
and the hydroxamic enzymes demonstrates that the modification only
slightly raises the apparent K
Another interpretation of the data obtained
might be that Asp
The results obtained point to the importance of the
negatively charged carboxyl at position 5 and confirm the suggestion
that the loss of activity after selective Asp
Reaction conditions and
calculations were the same as for Fig. 1. UPase (0.18 µM)
was preincubated with the ligands for 10 min in 0.05 M Tris-HCl, pH 7.1, after which WRK was added. The reaction mixtures
were incubated at 20 °C, and 20-µl aliquots were assayed for
UPase activity. ND, not determined.
Thanks are due to A. S. Mironov for the UPase
overproducing strain and to T. A. Egorov for peptide sequencing. We are
indebted to A. V. Galkin for helpful suggestions and for improving the
English version of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
= 0.07
mM) with subsequent modification of a carboxyl
( k
= 1.2 min
). Neither
substrate alone protected uridine phosphorylase from inactivation. The
presence of phosphate did not affect the K
and k
values. The addition of uracil or
uridine led to a significant increase of both K
(to 2.5 or 2.1 mM, respectively) and k
(to 6.1 or 4.8 min
, respectively) values.
Thus, WRK could react in accordance with slow (high affinity) and fast
(low affinity) mechanisms. Combined addition of phosphate and uracil
completely protected uridine phosphorylase. Tryptic digestion yielded a
single modified peptide
(Ser
-Asp(WRK)-Val-Phe-His-Leu-Gly-Leu-Thr-Lys
).
Treatment of the modified enzyme with hydroxylamine led to removal of
the bulky WRK residue and replacement of the Asp
carboxyl
by a hydroxamic group. The enzyme thus obtained recovered about 10% of
initial specific activity, whereas its substrate binding ability
changed only moderately; the K
values for
phosphate and uridine were changed from 5.1 and 0.19 mM (or
7.3 and 0.14 mM according to Leer et al. (Leer, J.
C., Hammer-Jespersen, K., and M. Schwartz (1977) Eur. J. Biochem. 75, 217-224)) to 22.6 and 0.12 m
M, respectively.
The hydroxamic enzyme had higher thermostability than the native
enzyme. The results obtained demonstrated the importance of the
carboxyl at position 5. The loss of activity after selective group
replacement is due to impaired stabilization of the transition state
rather than to a decline in substrate affinity or change of the active
site structure.
(
)
(EC
2.4.2.3) from Escherichia coli catalyzes reversible
phosphorolysis of uridine to ribose 1-phosphate and uracil (Leer et
al.,1977; Vita et al.,1986) and is
involved in pyrimidine nucleoside breakdown and utilization as carbon
and energy sources. The UPase structural gene has been cloned in
multicopy plasmids, and an overproducing strain has been isolated
(Brykun et al., 1989). The 253-amino acid sequence of E.
coli UPase is known (Walton et al., 1989); the enzyme
molecule consists of six identical subunits (Cook et al.,
1987; Tsuprun et al., 1991). To date, high resolution x-ray
structures have been established for the purine (Walter et
al., 1990) and thymidine phosphorylases (Ealick et al.,
1990), and preliminary results of UPase x-ray analysis have been
reported (Cook et al., 1987; Mikhailov et al., 1992).
2 mechanism with phosphate activation due to its
desolvation in the enzyme active site.
Chemicals
Woodward's reagent K (WRK),
Tris, TPCK-trypsin, endoproteinase from Staphylococcus aureus strain V, uridine (Urd), uracil (Ura),
5,5`-dithiobis(2-nitrobenzoate), trifluoroacetic acid, SDS, and
acrylamide were obtained from Sigma. Sephadex G-50M was from Pharmacia
Biotech Inc. Acetonitrile was from Aldrich. All other chemicals were of
reagent or analytical grade and were used without further purification.
Enzyme Preparation and Assay
UPase was isolated
from the E. coli K12 overproducing strain (kindly provided by
Prof. A. S. Mironov, GNII Genetika, Moscow) as described previously
(Mikhailov et al., 1992). The resulting enzyme was homogeneous
in SDS-polyacrylamide gel electrophoresis (Laemmli, 1970), had a
specific activity of 100-120 units/mg (25 °C), and an
A/ A
ratio of
1.74-1.78. The UPase activity was assayed with a Graficord UV-240
spectrophotometer (Shimadzu, Japan) according to Magni (1978). Protein
concentration was determined by the method of Bradford (1976) with
bovine serum albumin as a standard or from absorbance at 280 nm (Leer
et al., 1977).
Reaction of WRK with E. coli Uridine
Phosphorylase
Fresh WRK solutions (1.0-10.0 mM)
in cold 0.1 mM HCl were made for each experiment. The enzyme
(0.16 µM) was incubated at 20 °C with varying WRK
concentrations (10-200 µM) in 50 mM
Tris-HCl, pH 7.1. The control sample was incubated under the same
conditions without the reagent. At various times (0-5 min),
aliquots of 5-20 µl were withdrawn from both incubation
mixtures to measure the activity. Protection against inactivation was
assessed with the following substrates and products added to the enzyme
solution 10 min before WRK was added: 100 mM P, 5
mM Urd, 5 mM Ura, and 5 mM Urd plus 100
mM P
or 5 mM Ura plus 100 mM
P
. Stoichiometry of WRK incorporation into UPase was
measured after exposing the enzyme (36 µM) to WRK
(10-200 µM) for 5 min. Modified UPase was isolated
by gel filtration on a Sephadex G-50M column in 50 mM
Tris-HCl, pH 7.1. The number of modified carboxyls was estimated from
the peak absorbance (about 350 nm) but using a published extinction
coefficient of 7000 M
cm
at 340 nm (Sinha and Brever, 1985). Free SH groups of the
modified enzyme were titrated with 5,5`-dithiobis(2-nitrobenzoate) by
the method of Ellman (1959) in the presence of 6 M guanidine
HCl. The residual activity of the enzyme samples obtained was
determined as above and expressed as a percentage of initial activity.
Enzyme samples treated in a similar manner in the presence of an
equilibrium substrate-product mixture (addition of 5 mM Urd
and 100 mM phosphate) served as control.
Modified Enzyme Digestion and Peptide
Isolation
The enzyme modified with WRK (5 mg) was lyophilized
and dissolved in 0.1 ml of 6 M guanidine HCl; then 0.9 ml of
100 mM Tris-HCl, pH 7.5, was added. Modified UPase was
incubated with TPCK-trypsin or S. aureus proteinase V(1:50, w/w) at 37 °C for 6 h. The digest was centrifuged, and
the supernatant was lyophilized, dissolved in a small volume of
distilled water, and applied to a Zorbax PEP RP/1 (DuPont NEN) column
equilibrated with solvent A (20 mM sodium phosphate, pH 6.0).
HPLC was carried out using a Gilson model 704 apparatus, and absorbance
at 215 and 340 nm was detected with a model 116 detector (Gilson) and
Lambda-Max LC model 481 Spectrophotometer (Waters Co.). Peptides were
eluted with a linear gradient of solvent B (20 mM sodium
phosphate, pH 6.0, in 60% acetonitrile, 40% H
O (v/v))
(System 1) from 0 to 50% (1%/min). The flow rate was 1.0 ml/min. Peaks
absorbing at both 215 and 340 nm were collected and lyophilized. Then
the dry material was dissolved in a minimal volume of distilled water
and injected into the same column equilibrated with 0.1% (v/v)
trifluoroacetic acid in H
O (System 2). Rechromatographed
peptides were eluted with a 0-40% gradient of acetonitrile
(1%/min). The flow rate was 1.0 ml/min. All separations were performed
at room temperature.
Further Fragmentation of Modified
Peptides
Approximately 10 nmol of the WRK-modified peptides
obtained from HPLC separations in System 1 were lyophilized and
redissolved in 0.1 ml of 50 mM Tris-HCl at pH 7.5. The Vand tryptic peptides were further digested with TPCK-trypsin or
S. aureus V
proteinase, respectively (see above).
The digests obtained were resolved in System 2 as described above.
Modified peptide sequencing was carried out in the laboratory of Prof.
T. A. Egorov (Institute of Molecular Biology, Russian Academy of
Science, Moscow) using an Knauer model 910 sequencer.
Selective Substitution of Hydroxamate for Carboxylate in
the UPase Active Site
The WRK-modified enzyme (5 mg; residual
activity less than 1%) was incubated with 1.5 M hydroxylamine,
pH 6.8, for 1 h at 25 °C. A control sample of native UPase was
incubated with hydroxylamine under the same conditions. At various
times (0-60 min), aliquots of 5-20 µl were withdrawn
from both samples. Hydroxamic UPase was isolated by gel filtration on a
Sephadex G-50M column in 50 mM Tris-HCl, pH 7.1. The protein
was again consecutively treated with WRK and hydroxylamine and purified
as above. The number of modified carboxyl groups and the restitution of
activity were determined (see above).
Comparison of Native and Hydroxamic UPases
The
Kfor phosphate and uridine and
V
were determined at pH 7.1 at 25 °C and
compared with the literature data (Leer et al., 1977; Vita
et al., 1986). The thermostability of native and hydroxamic
enzymes was assessed by the apparent inactivation rate constants at 55
and 60 °C without ligands and with 5 mM Urd or 100
mM P
, pH 7.1. The Enzfitter program (version 1.05
(H)) was used to analyze the obtained data. Data are the means of at
least two separate experiments.
UPase Inactivation with WRK
Incubation of UPase
with WRK at pH 7.1 resulted in a time- and concentration-dependent loss
of enzymic activity (Fig. 1 A). Semilog plots of residual
activity versus time at various concentrations of WRK are not
linear after 4 min of incubation, probably because of the reagent
decomposition (Petra, 1971). A plot of the observed rate constants
( k) versus reagent concentration (over
the range 10-200 µM; Fig. 1 B) shows
saturation kinetics. The reagent can therefore be thought to bind
reversibly to the enzyme before covalent modification takes place,
is the apparent E*I
dissociation constant, and k
is the intrinsic rate
constant for covalent modification of UPase.
Figure 1:
Inactivation of UPase by WRK.
A, UPase (0.16 µM) was treated with 10 (),
20 (▾), 50 (
), 100 (
), and 200 (
)
µM WRK as described under ``Experimental
Procedures.'' At various time intervals, aliquots were withdrawn
and assayed for residual UPase activity. UPase incubated in the absence
of WRK lost no activity under the same conditions. B,
concentration dependence of the pseudo first-order rate constant,
k
.
The observed
inactivation rate constant ( k) can be expressed
as k
= k
/(1 +
K
/[I]) or 1/ k
= 1/ k
+
K
/ k
[I].
Fig. 1B shows the reciprocal plot of k
versus WRK concentration. This plot yields
K
= 0.07 ± 0.011
mM and k
= 1.2 ± 0.13
min
.
Stoichiometry of the Reaction of UPase with
WRK
The absorption spectrum of the modified enzymes has a band
at about 350 nm, consistent with an enol ester (formed in the reaction
of WRK with a carboxyl; Fig. 2 A) but with a red shift
(see ``Discussion''). The extent of inactivation correlated
with the reagent incorporation, and the data in Fig. 2 B indicate that the enzyme is inactivated upon modification of less
than two carboxyls in its subunit. Under an excess of enzyme (36
µM UPase and 20 µM WRK), 30% inactivation is
concomitant with incorporation of 0.4 mol of reagent/mol of subunit.
Complete inactivation with WRK is not accompanied by a decrease in free
sulfhydryl groups (three/UPase subunit) (Walton et al., 1989;
Bose and Yamada, 1974).
Figure 2:
A, absorption spectra of UPase partly
modified by WRK (residual activity, 70%). B, correlation
between enzyme activity and residue modification by WRK. Samples of
UPase (36 µM) were incubated with 20-200
µM WRK for 5 min in 0.1 M Tris-HCl, pH 7.1.
Modified enzyme was isolated by gel filtration, and reagent
incorporation and residual enzymic activities were
determined.
Effect of Substrates and Products on the Reaction of
UPase with WRK
An attempt was made to infer the place of reagent
binding in the UPase active site from the influence of substrates and
products on enzyme inactivation. The kin the
presence of various ligands is shown in . The inactivation
of UPase with 0.2 mM WRK was not prevented by preincubation of
the enzyme with any ligand used alone. However, almost complete
protection was observed upon simultaneous addition of 5 mM
uracil and 100 mM phosphate or 5 mM uridine and 100
mM phosphate (equilibrium substrate-product mixture)
().
) combination, the dependence of the
k
on the concentration of one ligand (P
or Ura) at a saturating concentration of the other one was
determined (Fig. 3). The data obtained suggest that the
synergistic protective effect is only due to simultaneous filling of
the corresponding subsites in the UPase active site
( K
= 7.3 and 0.24 mM,
respectively) (Leer et al., 1977). It should be noted that
filling of ribose and phosphate subsites with deoxyribose 1-phosphate
or ribose and uracil subsites with Urd provided no protection against
inactivation with WRK ().
Figure 3:
Effect of one ligand (P or
Ura) on the inactivation rate constant ( k
) at a
saturating concentration of the second one. A, dependence of
the pseudo first-order rate constant ( k
) with
0.2 mM WRK on uracil concentration ([P
]
= 100 mM throughout). B, dependence of the
k
with 0.2 mM WRK on phosphate
concentration ([Ura] = 5.0 mM throughout).
Conditions were the same as described in the legend to Fig. 1. Ligands
were added 10 min before WRK.
Furthermore the
Kand k
values
() were determined under the conditions of saturation with
P
, Urd, and Ura (). The binding of phosphate in
the active site of UPase does not affect the inactivation kinetics. The
binding of uridine and uracil strongly diminishes the enzyme affinity
for the inhibitor ( K
= 2.1 and 2.5
mM, respectively) but significantly increases the
k
(from 1.2 to 4.8 and 6.1 min
,
respectively). These results probably reflect the existence of two
mechanisms of WRK interaction with UPase: high affinity (slow) and low
affinity (fast). Comparison of the values obtained under saturation
with Urd and Ura also confirms the suggestion that binding at the
ribose subsite does not appreciably influence the reagent affinity and
the inactivation rate ().
Modified Peptide Isolation and
Identification
After digestion with trypsin or S. aureus proteinase V, the resulting peptides were initially
resolved by reversed-phase HPLC at pH 6.0 (System 1). Under these
conditions the enol ester is stable enough for the modified peptide to
be isolated. The elution profile of the tryptic peptides
(Fig. 4 A) showed a single peak absorbing at both 215 and
340 nm. When the V
peptides were eluted in an analogous
manner from the same column, a single peak of absorbance at 340 nm was
eluted 3 min later (data not shown). The tryptic and V
peptides were further digested with proteinase V
and
trypsin, respectively, and analyzed by HPLC using System 2. Only the
V
peptide showed a change in retention time after
additional trypsin treatment (Fig. 4 B) (coincidence with
tryptic and tryptic-plus-V
peptides). These results
suggested that the tryptic peptide did not contain V
cleavage sites. After rechromatography of the tryptic modified
peptide (Fig. 4 B), the amino acid sequence was
determined by automatic Edman degradation. The first six residues of
the peptide coincided with those in the tryptic peptide
Ser-Asp-Val-Phe-His-Leu-Gly-Leu-Thr-Lys occupying positions 4-13
in the UPase (Walton et al., 1989). Thus, the only
dicarboxylic amino acid, Asp
, was modified with WRK.
Because the WRK residue was eliminated during Edman degradation,
Asp
was determined as a phenylthiohydantoin. The modified
peptide was isolated and sequenced twice from different specimens of
modified UPase.
Figure 4:
HPLC resolution of a tryptic digest of
WRK-inhibited UPase. A, modified UPase (5 mg) was digested
with TPCK-trypsin as described under ``Experimental
Procedures.'' The digest was injected onto a reversed-phase HPLC
column (Zorbax PEP RP/1; DuPont NEN) equilibrated with 20 mM
sodium phosphate, pH 6.0. Peptides were eluted in System 1. For details
see ``Experimental Procedures.'' Absorbance was measured at
340 nm corresponding to enol ester. The single peak of absorbance at
340 nm was collected and lyophilized. B, rechromatography of
modified peptides in System 2. a, tryptic peptide; b,
tryptic peptide treated with proteinase V; c,
V
peptide; and d, V
peptide treated
with trypsin. For details see ``Experimental
Procedures.''
Production and Properties of UPase with Selective
Substitution of Asp
The WRK residue in the active center may cause
steric obstacles resulting in complete enzyme inactivation; therefore
the possible role of AspCarboxyl with
Hydroxamate
in the mechanism of the enzymatic
uridine phosphorolysis remains unclear. It is known that the enol ester
formed upon selective modification of the Asp
carboxyl by
WRK readily reacts with some nucleophilic agents that substitute for
the bulky WRK residue. Thus after reaction with hydroxylamine the
Asp
carboxyl proves to be selectively replaced by a
hydroxamic group. In this case the possible steric conflicts in the
active site are negligible, because only two atoms (NH) are added in
comparison with the initial structure (Fig. 5). The native enzyme
is not inactivated upon incubation with hydroxylamine. To ensure
completeness of the reaction, consecutive treatment of the enzyme with
WRK and hydroxylamine was done twice. The control sample was treated in
the presence of an equilibrium mixture of substrates and products of
the UPase reaction. The hydroxamic UPase thus obtained retained about
10% of the initial activity (the control sample retained about 75%
activity and exhibited the same properties as native UPase). The
increase in specific activity of the modified enzyme (from <1 to
about 10%) observed upon hydroxylamine treatment and accompanied by a
decline in absorbance at 350 nm appears to reflect the removal of the
WRK residue. Both native and hydroxamic UPase preparations demonstrated
equal mobility in SDS-polyacrylamide gel electrophoresis in the
presence or absence of 2-mercaptoethanol. However, they demonstrated
quite different behavior with respect to inactivation by WRK. With the
hydroxamic enzyme the inactivation was 10 times slower and probably
reflected nonspecific interaction of the agent with other carboxyls
(). Thus it could be suggested that selective replacement
of an essential carboxyl has been achieved. To ascertain the cause of
the observed differences in specific activities, the V
and K
values with individual
substrates were determined (). Although the
K
values for native and hydroxamic UPases
differ only slightly, the maximal rate for the modified enzyme is less
than one-tenth of that for the native one. Selective group replacement
at Asp
results in a marked increase in enzyme
thermostability (), so that after a long enough incubation
the residual specific activity of the hydroxamic enzyme may exceed that
of the native one by an order of magnitude.
Figure 5:
Interaction of WRK with Asp in
the UPase active center and its subsequent replacement with
hydroxamate. Percentages correspond to specific activity relative to
the native enzyme. For details see ``Experimental
Procedures.''
= 0.07 ± 0.01 mM
and k
= 1.2 ± 0.1
min
. The band at 350 nm appearing in the enzyme
spectrum upon modification with WRK (Fig. 2 A) is
indicative of an enol ester arising from interaction between WRK and a
carboxyl, with allowance for the red shift recently observed in an
analogous study with fluorescein 5`-isothiocyanate (Komissarov et
al., 1994b). Hence we can conclude that UPase is inactivated by
modification of a dicarboxylic amino acid. There are 28 free carboxyls
in each UPase subunit (Walton et al., 1989), and WRK can also
react with amino and sulfhydryl groups. However, the avidity of WRK for
the active site permits unambiguous identification of the target
residue. Exposure of the enzyme to a substoichiometric amount of WRK
(Fig. 2 B) results in 30% inactivation upon modification
of about 0.4 mol of residue/mol of subunit; quantitation of the
spectral changes evoked by the WRK treatment in the absence and in the
presence of protective agents (not shown) also testifies to
modification of a single essential carboxyl. Again, no appreciable
decrease in the number of free SH groups was found for the WRK-modified
enzyme.
does not protect the enzyme from
inactivation by WRK (). Only when the uracil and the
phosphate subsites are occupied simultaneously is the enzyme almost
completely protected ( and Fig. 3). The presence of
an equilibrium mixture of substrates and products of the enzymatic
reaction also provides nearly complete protection against WRK.
Concurrent filling of the ribose subsite has no effect on the
inactivation, indicating that WRK perhaps does not interact with this
subsite. Taken alone, P
does not change the
K
and k
values,
whereas Ura or Urd markedly diminish the inhibitor binding
().
value becomes comparable with those in
I; hence we may conclude that the marked selectivity of
WRK toward UPase is mainly due to interactions at the uracil subsite.
according to the sequence of Walton et al. (1989).
carboxyl with a hydroxamic group ( Fig. 5and
). Inactivation of the resulting enzyme by WRK is an order
of magnitude slower () and probably reflects nonspecific
interactions.
for
P
and Urd but significantly diminishes the
V
(). It appears that the Asp
carboxyl is not crucial for substrate binding but is dynamically
involved in catalysis.
takes part in maintaining the native
enzyme conformation. In such a case, however, the modification should
have impaired the stability of hydroxamic UPase as compared with the
control; instead we see that the thermostability of the modified enzyme
is substantially enhanced (). Thus we believe that the
Asp
residue is essential for the catalytic step proper
rather than for substrate binding or conformation. On the other hand,
the hydroxamic substituent may afford additional hydrogen bonds;
because the amino-terminal part of the UPase subunit is probably
involved in active site formation, this may have significant structural
and conformational effects and be the cause of enhanced enzyme
thermostability.
modification
is due to impaired stabilization of the transition state rather than to
a decrease in substrate affinity. The suggestion above is in agreement
with the results on glycosidases and glycosyltransferases. It was
consistently shown that in these enzymes a carboxyl group mediates
glycosyl transfer (Mooser et al., 1991). Notwithstanding,
additional studies including high resolution x-ray analysis of the
enzyme-substrate complex and site-directed mutagenesis are clearly
needed to get molecular insight into the enzymatic phosphorolysis of
uridine.
Table: Effects of substrates, products, and their
analogs on inactivation rate constant (k)
and K
and k
values (Scheme 1)
Table: Properties of the
native and hydroxamic enzymes
Table: Kinetic
parameters of inactivation of some enzymes with WRK
, endoproteinase V
from S.
aureus; WRK, Woodward's reagent K.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.