©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Complete Inactivation of Escherichia coli Uridine Phosphorylase by Modification of Asp with Woodwards Reagent K(*)

Andrey A. Komissarov (§) , Darya V. Romanova , Vladimir G. Debabov

From the (1) State Scientific Centre of Russian Federation GNII Genetika, 1st Dorozhny 1, Moscow, 113545, Russia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Woodward's reagent K (WRK) completely inactivated Escherichia coli uridine phosphorylase by reversible binding in the active site ( K= 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 Kand kvalues. 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 Aspcarboxyl by a hydroxamic group. The enzyme thus obtained recovered about 10% of initial specific activity, whereas its substrate binding ability changed only moderately; the Kvalues 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.


INTRODUCTION

Uridine phosphorylase (UPase)() (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).

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 S2 mechanism with phosphate activation due to its desolvation in the enzyme active site.

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.


EXPERIMENTAL PROCEDURES

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/ Aratio 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 Por 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 Mcmat 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% HO (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 HO (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 Vproteinase, 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 Vwere 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.


RESULTS

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,

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, Kis the apparent E*I dissociation constant, and kis 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) ().

To examine the protection mechanism of the product (Ura) and substrate (P) combination, the dependence of the kon the concentration of one ligand (Por 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 kwith 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 kvalues () 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 Vpeptides 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 Vpeptides were further digested with proteinase Vand trypsin, respectively, and analyzed by HPLC using System 2. Only the Vpeptide showed a change in retention time after additional trypsin treatment (Fig. 4 B) (coincidence with tryptic and tryptic-plus-Vpeptides). These results suggested that the tryptic peptide did not contain Vcleavage 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, Aspwas 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, Vpeptide; and d, Vpeptide treated with trypsin. For details see ``Experimental Procedures.''



Production and Properties of UPase with Selective Substitution of AspCarboxyl with Hydroxamate

The WRK residue in the active center may cause steric obstacles resulting in complete enzyme inactivation; therefore the possible role of Aspin the mechanism of the enzymatic uridine phosphorolysis remains unclear. It is known that the enol ester formed upon selective modification of the Aspcarboxyl by WRK readily reacts with some nucleophilic agents that substitute for the bulky WRK residue. Thus after reaction with hydroxylamine the Aspcarboxyl 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 Vand Kvalues with individual substrates were determined (). Although the Kvalues 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 Aspresults 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.''




DISCUSSION

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= 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.

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 Kdoes 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, Pdoes not change the Kand kvalues, whereas Ura or Urd markedly diminish the inhibitor binding ().

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 Kvalue 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.

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 Aspaccording to the sequence of Walton et al. (1989).

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 Aspcarboxyl with a hydroxamic group ( Fig. 5and ). Inactivation of the resulting enzyme by WRK is an order of magnitude slower () and probably reflects nonspecific interactions.

Comparison of the kinetic parameters for the native and the hydroxamic enzymes demonstrates that the modification only slightly raises the apparent Kfor Pand Urd but significantly diminishes the V(). It appears that the Aspcarboxyl is not crucial for substrate binding but is dynamically involved in catalysis.

Another interpretation of the data obtained might be that Asptakes 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 Aspresidue 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.

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 Aspmodification 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)

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.


  
Table: Properties of the native and hydroxamic enzymes


  
Table: Kinetic parameters of inactivation of some enzymes with WRK



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, 200158, M-121 Medical Sciences Bldg.-DC008.00, University of Missouri, Columbia, MO 65212. Tel.: 314-882-1493; Fax: 314-884-4597; E-mail: Andrey_A._Komissarov_at_MU-BNEM-PO1@mucmail.missouri.edu.

The abbreviations used are: UPase, uridine phosphorylase (EC 2.4.2.3) from E. coli; HPLC, high performance liquid chromatography; TPCK-trypsin, L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin; V, endoproteinase Vfrom S. aureus; WRK, Woodward's reagent K.


ACKNOWLEDGEMENTS

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.


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