A reexamination of the substrate utilization of 2-thioorotidine-5′ -monophosphate by yeast orotidine-5′ -monophosphate decarboxylase

Article abstract of DOI:10.1006/bioo.2001.1201

A potential alternate substrate for orotidine-5′-monophosphate decarboxylase, 2- thio-orotidine-5′-monophosphate, was synthesized enzymatically and purified by a modification of a previous account (K. Shostak, and M. E. Jones 1992, Biochemistry 31, 12155-12161). Characterization of the product was confirmed by mass spectrometry, ³¹P NMR, and utilization by orotate phosphoribosyltransferase in the direction of pyrophosphorolysis. The previous work probably did not result in the purification of the desired compound, as evidenced by our observation of 2-thioOMP's sensitivity to high temperature, as used previously. Using a very sensitive HPLC assay for the potential decarboxylated product 2-thioUMP, no measurable activity of ODCase toward the alternate substrate was observed, representing a decarboxylation rate decreased by 10^-7 from the k^cat for ODCase toward OMP. Additionally, 2-thioOMP effects no inhibition of ODCase decarboxylation of OMP at a concentration of 50 μM, indicating a poor ability to bind to the ODCase active site. The results bear implications for proposed mechanisms for catalysis by ODCase.

Full text of DOI:10.1006/bioo.2001.1201

Bioorganic Chemistry 29, 96–106 (2001)
doi:10.1006/bioo.2001.1201, available online at http://www.idealibrary.com on
A Reexamination of the Substrate Utilization of 2-Thioorotidine-
5Ј-monophosphate by Yeast Orotidine-5Ј-Monophosphate
Decarboxylase
Jeffrey A. Smiley,*,1 Kelly M. Hay,† and Bruce S. Levison‡
*Department of Chemistry and †Department of Biological Sciences, and the
‡Center for Biomedical and Environmental Research, Youngstown State University,
Youngstown, Ohio 44555
Received October 6, 2000
A potential alternate substrate for orotidine-5Ј-monophosphate decarboxylase, 2- thio-orotid-
ine-5Ј-monophosphate, was synthesized enzymatically and purified by a modification of a
previous account (K. Shostak, and M. E. Jones 1992, Biochemistry 31, 12155–12161). Character-
ization of the product was confirmed by mass spectrometry, 31P NMR, and utilization by orotate
phosphoribosyltransferase in the direction of pyrophosphorolysis. The previous work probably
did not result in the purification of the desired compound, as evidenced by our observation of
2-thioOMP’s sensitivity to high temperature, as used previously. Using a very sensitive HPLC
assay for the potential decarboxylated product 2-thioUMP, no measurable activity of ODCase
toward the alternate substrate was observed, representing a decarboxylation rate decreased by
10Ϫ7 from the kcat for ODCase toward OMP. Additionally, 2-thioOMP effects no inhibition of
ODCase decarboxylation of OMP at a concentration of 50 ␮M, indicating a poor ability to
bind to the ODCase active site. The results bear implications for proposed mechanisms for
catalysis by ODCase.
᭧ 2001 Academic Press
Recent protein crystal structures (1–4) have provided new insights into the catalytic
mechanism of OMP decarboxylase (ODCase),2 the enzyme with the highest measured
catalytic proficiency (5). Originally, the catalytic mechanism of ODCase was presumed
to proceed through a protonation step at O2 preceding decarboxylation (Fig. 1),
yielding a zwitterionic intermediate, based on the observation of rates of uncatalyzed
decarboxylation of model compounds (6). Support for this mechanism arose from
enzyme inhibition (7) and isotope effect studies (8–10). A lysine residue (Lys93 of
the yeast enzyme), common to all protein sequences of ODCases from many organ-
isms, was found to be critical for catalysis and postulated as the general acid group
1 To whom correspondence should be addressed at the Department of Chemistry, Youngstown State
University, One University Plaza, Youngstown, OH 44555. Fax: (330) 742-1579. E-mail: jasmiley@
cc.ysu.edu.
2 Abbreviations used: OMP, orotidine-5Ј-monophosphate; ODCase, OMP decarboxylase; 2-thioOMP,
2-thioorotidine-5Ј-monophosphate; Lys93, residue 93 (lysine) in the yeast ODCase sequence; OPRTase,
orotate phosphoribosyltransferase; PRPP, 5-phosphorylribose-1-pyrophosphate.
96
0045-2068/01 $35.00
Copyright ᭧ 2001 by Academic Press
All rights of reproduction in any form reserved.

NEGLIGIBLE INTERACTION OF ODCase WITH 2-thioOMP
97
FIG. 1. Proposed mechanism of ODCase catalysis by O2 protonation. Several resonance structures
are possible for distribution of charges in the intermediates; previous depictions have shown isolation of
+
the positive charge at N1. Enz-NH3 , lysine side chain at active site (Lys 93 of the yeast enzyme).
protonating the substrate (11). Substitution of sulfur for oxygen at the 2-carbonyl
position apparently resulted in a substrate analog, 2-thioorotidine-5Ј-monophosphate
(2-thioOMP), that was incapable of undergoing catalysis (12).
The four crystal structures (1–4) showed an orientation for the inhibitors with C6,
rather than C2, aligned with the invariant lysine residue. A mechanism proposed from
this active-site structure (1,3,4) suggests that this lysine side chain donates a proton
to C6 of OMP to directly replace the departing carboxylate (Fig. 2), invoking no
critical involvement of the substituent at C2.
In light of the new mechanism proposed, and to extend our previous finding that
2-thioUMP has essentially the same inhibition constant as UMP (13), a reexamination
of the substrate utilization of 2-thioOMP seemed warranted. Possibly, the enzymatic
synthesis method used previously (12) yielded a compound other than the anticipated
product 2-thioOMP. We have synthesized 2-thioOMP from 2-thioorotate, 5-phospho-
rylribose-1-pyrophosphate (PRPP) and orotate phosphoribosyltransferase (OPRTase),
and devised an HPLC assay for detection of 2-thioUMP which could be generated
from the decarboxylation by ODCase. We have found 2-thioOMP to be a fairly
unstable compound and suspect that the compound isolated previously (12) was likely
an undesired product of this instability. However, the 2-thioOMP we have synthesized
and characterized does not undergo enzymatic decarboxylation and does not bind to
ODCase with an affinity even remotely similar to that for OMP, calling again into
question the role of the 2-carbonyl group of the substrate during catalysis.

98
SMILEY, HAY, AND LEVISON
C
FIG. 2. Proposed mechanism of ODCase catalysis by C6 protonation. The pyrimidine ring is rotated
180Њ about the glycosidic bond from the structure in Fig. 1. The crystal structure of ODCase and UMP
(1) shows proximity of the active-site lysine with C6; this feature of the active-site structure is shown
in C.
MATERIALS AND METHODS
2-Thioorotate, previously purchased from Aldrich (12), is not currently commer-
cially available, and was instead synthesized from thiourea (Sigma) and oxaloacetate
monoethyl ester (from the diethyl ester, Sigma) according to a previous account (14).
OPRTase was a generous provision from the laboratory of Dr. Charles Grubmeyer,
Temple University. 2-Thiouracil and PRPP were from Sigma. OMP and [carboxyl-
14C]OMP were synthesized according to the modified procedure (9) of the original
synthesis (15). Yeast ODCase was isolated as the predominant protein eluting from
a column of Affi-Gel Blue affinity chromatography resin, as described previously (16).
2-thioOMP was enzymatically synthesized as described previously (12), with the
following modifications. Instead of terminating the reaction by boiling, the reaction
was quenched by the addition of ethanol to a final concentration of 50%, and precipi-
tated protein was removed by filtration. The solution was evaporated to a small
volume, and the products were separated by anion-exchange HPLC, using a Hamilton
PRP-X100 column (305 ϫ 7 mm) and the following elution profile, 0–10 min, 0–0.8
M NH4HCO3, pH 8.4, linear gradient; 10–25 min, isocratic 0.8 M NH4HCO3, pH
8.4. Products eluting from the column were detected by absorbance at 270 or 276
nm. 2-thioOMP was quantitated by absorbance at 276 nm, according to the extinction
coefficient (␧276 ϭ 11,000 MϪ1) obtainable from the isosbestic point in the conversion
of 2-thioorotate to 2-thioOMP demonstrated previously (12). NH3 present in HPLC

NEGLIGIBLE INTERACTION OF ODCase WITH 2-thioOMP
99
fractions of 2-thioOMP was assayed using Nessler’s reagent, with NH4HCO3 solution
as a standard. 2-thioUMP was enzymatically synthesized as before (13), using overpro-
duced uracil phosphoribosyltransferase from an Escherichia coli strain kindly provided
by Dr. Jan Neuhard, University of Copenhagen. 2-thioUMP was purified under the
same HPLC conditions as above.
Mass spectrometry of 2-thioOMP. An HPLC elution fraction corresponding to a
major peak arising in the 2-thioOMP enzymatic synthesis was subjected to analysis
on a Bruker Esquire mass spectrometer, under the following electrospray ionization
(ESI) conditions: ESI voltage, 4.0 kV; direct infusion; capillary temperature, 300ЊC;
anion detection; scan range, 50–2200 m/z; scan resolution, 0.6 m/z.
NMR analysis of 2-thioorotate and 2-thioOMP. Synthesized 2-thioorotate was ana-
lyzed by 13C NMR (proton decoupled), and 2-thioOMP analyzed by 31P NMR, using
a Varian Gemini 400 MHz NMR spectrometer. Samples of both compounds were
prepared in D2O, with NaOD added to the 2-thioorotate to increase solubility.
OPRTase-catalyzed pyrophosphorolysis of 2-thioOMP. Further structural analysis
of the suspected 2-thioOMP was assessed using the HPLC-purified material as a
substrate for the pyrophosphorolysis reaction catalyzed by OPRTase. Reaction condi-
tions were identical to the OPRTase reaction for 2-thioOMP synthesis, with the
exception of 40 ␮M 2-thioOMP and 200 ␮M sodium pyrophosphate replacing 2-
thioorotate and PRPP as substrates, and the exclusion of inorganic pyrophosphatase.
Products were separated under the same HPLC conditions as used for the purification
of 2-thioOMP.
Enzymatic assays of ODCase using OMP and 2-thioOMP. ODCase activity at
saturating OMP concentration (50 ␮M) was measured using a spectrophotometric
assay (17) with ⌬␧ ϭ Ϫ2250 MϪ1. Decarboxylation activity was measured in the
286
presence of 50 mM Tris-Cl, pH 8.0. For reactions containing 200 mM NH4HCO3, a
solution of 1 M Tris-Cl, pH 6.9, was added to give a final concentration of 50 mM
Tris and a final pH of 8.0. Activity was measured within the first minute of initiating
the reaction. Activities at subsaturating concentrations were measured using the 14C
displacement assay with [carboxyl-14C]OMP (18).
Activity of ODCase toward 2-thioOMP was measured by detection of 2-thioUMP
by HPLC, with the measured retention times for 2-thioOMP (10 min) and 2-thioUMP
(6.8–6.9 min) under the separation conditions described above. 2-thioUMP was quanti-
tated using ␧ ϭ 13,000 MϪ1. The concentration of 2-thioOMP was 50 ␮M, and
274
the ODCase concentration was increased to as high as 220 ␮g mLϪ1. Tris-Cl was
added to a final concentration of 50 mM, pH 8.0, and NH4HCO3 (from 2-thioOMP
HPLC fractions) was 200 mM. From 1.0-mL reactions, 100-␮L samples were taken
for HPLC analysis. In some samples, 2-thioUMP was included as a standard.
RESULTS AND DISCUSSION
Shostak and Jones (12) showed that the activity of OPRTase toward 2-thioorotate
and PRPP was consistent with formation of a nucleotide closely resembling OMP,
from the UV absorbance spectra of the enzymatic reaction. The OPRTase used in
this enzymatic synthesis has a high specificity for orotate versus other heterocycles,
due to hydrogen bonding of N3–O4 and the carboxylate of the natural substrate (19);
these strucural features of the pyrimidine substrate are unchanged in the thio analog.

100
SMILEY, HAY, AND LEVISON
The lack of activity of OPRTase toward 5-carboxyuracil (C. Grubmeyer, personal
communication), for example, indicates that the exact location of the carboxylate
group on the pyrimidine is critical for binding and catalysis, and formation of the
expected 2-thioOMP by this enzyme is quite predictable.
Our enzymatic synthesis of 2-thioOMP produced a major peak in the HPLC profile
emerging at 10 min,well before the elution of 2-thioorotate. This peak increased in
intensity with length of time of the reaction and presence of inorganic pyrophosphatase
to remove pyrophosphate (Fig. 3). The previous description of the enzymatic synthesis
of 2-thioOMP (12) reported that the nucleotide emerged from anion-exchange HPLC
after the starting compound. We thus subjected our product to further analysis.
13C NMR analysis of 2-thioorotate showed peaks with the following chemical shifts
and their tentative assignments: 181.7 ppm (C2), 176.2 ppm (C7), 174.9 ppm (C4),
161.1 ppm (C6), 104.0 ppm (C5). The pattern is similar to, but distinct from, the 13
C
signals from orotate, and no discernable traces of orotate signals in this preparation
were evident.
Mass spectrometry of the 10-min fraction revealed the following anionic masses:
339.0, 360.9, 383.0, and 405.0 (Fig. 4). The mass of 383.0 matches that for the
monoanion of 2-thioOMP. The mass of 405.0 matches that for the monoanion of 2-
thioOMP in which Na+ has replaced one dissociable proton. The two lower masses
are in agreement with the monoanion and Na+-substituted monoanion of 2-thioUMP,
suggesting decarboxylation within the conditions of the mass spectrometer. Analysis
FIG. 3. HPLC traces of 2-thioOMP synthesis. Enzymatic synthesis reactions with 2-thioorotate and
OPRTase are as described in Ref. (12), with the modifications described in the text. Chromatograms are
normalized so that the highest peak within each trace is assigned an absorbance of 1.0. A: 2-Thioorotate
only. B: 2-thioOMP enzymatic synthesis, 1 h at 30ЊC, without inorganic pyrophosphatase. C: 2-thioOMP
enzymatic synthesis, 1 h at 30ЊC, with 10 units inorganic pyrophosphatase. D: 2-thioOMP enzymatic
synthesis, 3 h at 30ЊC, with 10 units inorganic pyrophosphatase, with 2-thioUMP included as a reference
standard. The fractions from the peaks at 10 min were collected and analyzed. The peaks in B–D at 13
min arise in control reactions without OPRTase (data not shown), and are presumed to be indicative of
a contaminant in the PRPP.

NEGLIGIBLE INTERACTION OF ODCase WITH 2-thioOMP
101
FIG. 4. Mass spectra of synthesized 2-thioOMP (A) and OMP (B). The expected molecular weights
of the 2-thioOMP and OMP monoanions are 383 and 367, respectively. Origins of the additional peaks
in the spectra are detailed in the text.
of OMP, synthesized separately, gave major peaks of 367.10 and 323.38 (Fig. 4),
again suggesting decarboxylation within the conditions of the mass spectrometer.
31P NMR of 2-thioOMP gave a single signal of Ϫ3.2 ppm (phosphate reference
standard set at 0.0 ppm), resembling the value of Ϫ3 ppm (relative to phosphate)
reported for OMP (20). The single 31P NMR signal indicates only one nucleotide
present, with no isomerization or other undesired side reaction evident.
The isolated nucleotide was completely converted to 2-thioorotate in the OPRTase-
catalyzed pyrophosphorolysis reaction. The OPRTase pyrophosphorolysis reaction
resulted in the complete disappearance of the 10 min HPLC peak, and formation of
a 2-thioorotate peak at 22 min (Fig. 5). From the increased production of the HPLC

102
SMILEY, HAY, AND LEVISON
FIG. 5. Conversion of synthesized 2-thioOMP to 2-thioorotate via pyrophosphorolysis with OPRTase.
Lower trace: 3.3 nmol synthesized 2-thioOMP. Upper trace: 3.0 nmol total thiopyrimidine following
pyrophosphorolysis reaction with OPRTase. The portions of the HPLC traces between 11.5 and 19.5 min
contained no peaks and were omitted for conciseness.
fraction upon enhancing the OPRTase synthesis reaction conditions, mass spectromet-
ric analysis, 31P NMR analysis, and the ability of the isolated compound to act as an
OPRTase pyrophosphorolysis substrate, we conclude that the 10-min fraction from
HPLC purification does in fact contain 2-thioOMP, and that no unwanted side reactions
occur in our preparation.
The conflict with the relative retention times of 2-thioorotate and 2-thioOMP in
our study and the previous report (12) remained to be resolved. We observed retention
times for 2-thiouracil and 2-thioUMP of 17.5 and 6.8–6.9 min, respectively, and
assumed that similar but longer retention times would be found for 2-thioorotate
and 2-thioOMP. The longer retention times for pyrimidine bases, compared to their
respective nucleotides, on anion-exchange HPLC under these conditions are likely a
function of the increased hydrophobic interactions of the bases with the matrix of
the anion-exchange resin. We also observed that prolonged evaporation of the 2-
thioOMP solution, to remove NH4HCO3, resulted in new peaks with longer retention
times upon HPLC analysis of the evaporated material. Suspecting chemical instability
of the 2-thioOMP, we subjected a sample of purified nucleotide to a boiling water
bath for 3 min, speculating on the unspecified conditions for terminating the OPRTase

NEGLIGIBLE INTERACTION OF ODCase WITH 2-thioOMP
103
reaction in the previous report. HPLC analysis of the heat-treated sample showed
two peaks with longer retention times, indicating conversion of some of the 2-thioOMP
to uncharacterized products (data not shown). We suspect that one of these products
was isolated in the previous study.
Due to the observed instability of 2-thioOMP to evaporation for removal of
NH4HCO3, we evaluated the activity of ODCase toward this potential alternate sub-
strate in the presence of NH4HCO3 that eluted with the 2-thioOMP, and compared
the results to ODCase activity toward OMP in the presence of the same amount of
NH4HCO3. In the spectrophotometric assay of ODCase activity toward OMP (at
saturating OMP concentration), activity in 200 mM NH4HCO3, pH 8.0, is modestly
reduced to about 80% of its maximal activity in the absence of NH4HCO3.
The HPLC assay of ODCase activity was sufficiently sensitive to detect traces of
UMP (retention time ϭ 6.3–6.5 min) and 6-azaUMP (retention time ϭ 7.6–7.7 min)
present in the ODCase preparation, that had not been removed by dialysis during
enzyme purification. The measured amounts of these nucleotides that were present
in the assay of activity toward 2-thioOMP were well below their respective Ki values
(7) and thus effected only a negligible degree of inhibition. The peaks for UMP and
6-azaUMP afforded convenient markers for the detection of 2-thioUMP that could
potentially be produced from ODCase and 2-thioOMP.
Assays of ODCase activity toward 2-thioOMP showed that this substrate analog
is at best an extremely poor substrate. In 1.0-ml assays with 220 ␮g ODCase and 50
␮M 2-thioOMP, the 2-thioOMP HPLC peak remained unchanged and no 2-thioUMP
was detectable after 120 min. Using synthesized 2-thioUMP as a standard in a control
HPLC analysis, the amount of 2-thioUMP produced under this reaction condition,
FIG. 6. HPLC traces of ODCase assays with 2-thioOMP. The portions of the chromatograms from
6 to 8 min show peaks corresponding to UMP (6.3–6.5 min), 2-thioUMP (6.8–6.9 min), and 6-azaUMP
(7.7–7.8 min). Reaction conditions are described in the text; 100-␮L volumes from 1.0-mL reactions are
analyzed. Chromatograms are normalized so that the highest peak within each trace is assigned an
absorbance of 1.0. A: 220 ␮g/mL ODCase plus 50 ␮M 2-thioOMP, t ϭ 0 min. B: 220 ␮g/mL ODCase
plus 50 ␮M 2-thioOMP, t ϭ 120 min. C: 220 ␮g/mL ODCase plus 50 ␮M 2-thioOMP plus 0.025 nmol
2-thioUMP, t ϭ 0 min.

104
SMILEY, HAY, AND LEVISON
possibly undetectable in the baseline noise, is estimated to be less than 0.10 nmol
(Fig. 6). The amount of conversion of 2-thioOMP to 2-thioUMP in this assay was
thus less than 3.8 ϫ 10Ϫ6 nmol minϪ1 ␮gϪ1, decreased from the kcat of ODCase
toward OMP (16) by a factor of 10Ϫ7.
Contrary to the previous report (12), 2-thioOMP exerts no inhibition of ODCase
activity toward OMP at concentrations in the range of Km for OMP. Assays of ODCase
activity with and without 50 ␮M 2-thioOMP are essentially identical (Fig. 7); thus
the Ki for this substrate analog is well over 100 ␮M. By contrast, the Km for OMP
in optimal conditions is 0.7 ␮M; under the conditions of high NH4HCO3 concentration,
the Km is raised to approximately 4 ␮M.
The striking result from thio-substitution studies is that OMP binding to the ODCase
active site is essentially eliminated by 2-thio substitution, whereas UMP binding to
the same active site is hardly affected by 2-thio substitution (13). In the four published
crystal structures (1–4), there appears to be little steric constraint at O2 of each
bound ligand, including O2 of UMP complexed to the Bacillus subtilis enzyme (1).
Additionally, Miller et al. (21) have shown that removal of the Gln215 side chain,
the only active-site functional group within proximity of O2 of the bound ligand, is
inconsequential for decarboxylase activity. It is predictable from these observations
that 2-thioUMP would have approximately the same affinity as UMP, as there appears
˚
to be ample accommodation for the larger CϭS group, which is typically 0.4 A longer
than a CϭO double bond (22). It is not evident, however, why 2-thioOMP does not
bind and undergo decarboxylation if it occupies the same orientation as UMP as seen
in the crystal structures, and that projected for OMP by the mechanism in Fig. 2.
OMP apparently binds to the ODCase active site in an orientation that does not
tolerate 2-thio-substitution, but UMP binds in an orientation that is indifferent to 2-
thio-substitution. A plausible explanation for this observation is that OMP binding
to ODCase is fundamentally different from UMP binding (when UMP is added to
the active site as a ligand as opposed to produced by decarboxylation). Possibly, OMP
FIG. 7. Assay of ODCase inhibition by 2-thioOMP. Assays, by the 14CO2-displacement method, were
carried out with (●) or without (ࡗ) 50 ␮M 2-thioOMP. All assays contained 500 mM NH4HCO3, since
this salt was present in HPLC fractions of 2-thioOMP. Velocity data are given in 1/(nmol minϪ1 ␮gϪ1).

NEGLIGIBLE INTERACTION OF ODCase WITH 2-thioOMP
105
binds at the active site in an orientation with the pyrimidine ring rotated 180Њ about
the glycosidic bond with respect to the inhibitors seen in the crystal structures (compare
Fig. 1A with Fig. 2C). The near symmetry of the pyrimidine rings of ODCase ligands,
especially 6-hydroxyuridine-5Ј monophosphate (BMP), has previously prompted spec-
ulation on the orientation of inhibitor and substrate binding (2,4,7,11). Binding of a
nearly symmetrical heterocyclic substrate analog in an orientation reversed from that
of the substrate has precedent in the binding of the pteridine ring of methotrexate to
dihydrofolate reductase (23). From the crystal structures of ODCase in complex with
inhibitors, an orientation of OMP with the pyrimidine ring rotated 180Њ about the
glycosidic bond (with respect to the binding of inhibitors) would align O2 for proton-
ation by Lys93, in accordance with the original mechanism.
If OMP binds with O2 oriented toward the active-site lysine (Fig. 1), the steric
constraints might be prohibitive for 2-thioOMP binding and catalysis, in accord with
the observations herein. The yeast enzyme (2) and the Escherichia coli enzyme (4)
˚
each show a 2.7 A distance between the terminal N of the active-site lysine and O6
of BMP (analogous to O2 of OMP in a 180Њ-rotated conformation). The enzyme’s
attempt to bind 2-thioOMP, using the mechanism in Fig. 1, would be affected by a
˚
decrease in this distance of 0.4 A compared to OMP, and this effect might well
account for the lack of binding and decarboxylation of 2-thioOMP. Certainly, the role
of O2 in OMP decarboxylation is different from that of O4, which can tolerate thio-
substitution (24).
It is intriguing to note that binding of OMP with the pyrimidine ring rotated 180Њ
with respect to the binding of the inhibitors in the crystal structures would result in
proximity of the carboxylate with the 5Ј-phosphate of the substrate (depicted in Fig.
1). The role of decarboxylases is, of course, to promote electron flow off of the
carboxylate and onto the remainder of the substrate, which presumably is stabilized
by other molecular features of the active site. This role appears to be fulfilled in part
by an active-site glutamate side chain in histidine decarboxylase (25). An anionic
phosphate, in this case part of the substrate rather than part of the enzyme, would be
perfectly suited for this task as well. This possible mechanistic feature is supported
by the observation of profound decreases in ODCase activity toward orotate and
orotidine (26, 27).
ACKNOWLEDGMENTS
We gratefully acknowledge the provisions of phosphoribosyltransferases from Dr. Charles Grubmeyer
and Dr. Jan Neuhard. ODCase purification by Lana Saleh (current address, Graduate Program, Biochemistry
and Molecular Biology, Penn State University) is acknowledged with gratitude. This work was supported
by Grant CC4350 from Research Corporation, and in part by the Youngstown State University Presidential
Academic Center for Excellence in Research, Center for Biotechnology, and by Grant 34650-B4 from
the American Chemical Society Petroleum Research Fund.
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