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Hydrogen isotope tracing in the reaction of orotidine-5′-monophosphate decarboxylase
ABB Archives of Biochemistry and Biophysics 412 (2003) 267–271 www.elsevier.com/locate/yabbi
Hydrogen isotope tracing in the reaction of orotidine-50-monophosphate decarboxylase Jeffrey A. Smiley,* Brian J. DelFraino, and Beth A. Simpson Department of Chemistry and Center for Biotechnology, Youngstown State University, One University Plaza, Youngstown, OH 44555, USA Received 19 November 2002, and in revised form 31 January 2003
Abstract The mechanism of the enzyme orotidine-50 -monophosphate decarboxylase (OMP decarboxylase, ODCase) is not fully characterized; some of the proposed mechanisms suggest the possibility of hydrogen rearrangement (shift from C5 to C6 or loss of H5 to solvent) during catalysis. In this study, we sought mechanistic information for the ODCase reaction by examining the extent of hydrogen exchange in the product uridine-50 -monophosphate, in combination with ODCase, at the H5 and H6 positions. In a subsequent experiment, partially deuterated OMP was prepared, and the extent of 2 H5 rearrangement or loss to solvent was examined by integration of 1 H nuclear magnetic resonance signals in the substrate and the resulting enzymatically decarboxylated product. The absence of detectable hydrogen exchange in these experiments limits somewhat the possible mechanisms for ODCase catalysis. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Orotidine-50 -monophosphate decarboxylase; Isotope labeling; Isotope exchange; Decarboxylation; Enzyme mechanism; NMR
Orotidine-50 -monophosphate decarboxylase (OMP decarboxylase, ODCase)1 has been the subject of heightened interest recently ([1–12]; review [13]). The original mechanism proposed for this enzyme reaction [14] involved proton donation to O2, forming a zwitterion intermediate (Fig. 1A). Various mechanistic studies have lent support to this model [15–20]. Four crystal structures of ODCases from different organisms were recently resolved [1–4], showing the C6 portion of the pyrimidine ring of bound ligands, rather than O2, facing the proposed proton-donating lysine side chain. These structures have led to the formulation of an electrophilic substitution mechanism (Fig. 1B) using protonation and direct CO2 elimination. Most recently, another mechanism has been proposed [21] in which C5 is protonated, leading to the formation of a different zwitterion (Fig. 1C). We were intrigued by an experimental detail from a very early report on ODCase [22]. Creasey and Hands* Corresponding author. Fax: 1-330-941-1579. E-mail address: [email protected] (J.A. Smiley). 1 Abbreviations used: OMP decarboxylase, ODCase, orotidine-50 monophosphate decarboxylase.
chumacher [22] prepared [5-3 H]OMP, decarboxylated this labeled substrate with ODCase, and analyzed the resulting tritiated UMP product using a chemical test with Br2 , designed to liberate 3 H specifically from the 5position of the pyrimidine. They found that only 70% of the 3 H label could be liberated from the resulting UMP and attributed this result to an incomplete 3 H release reaction. We have used the bromination of UMP and other uracil analogs in various syntheses and found the reaction to be virtually complete in a short time. Thus, we wondered whether the incomplete 3 H liberation might be a mechanistic clue and whether some of the 3 H originally present at C5 had been transferred to another molecular site. We considered that the proposed O2 protonation mechanism (Fig. 1A) might involve a rearrangement of some fraction of the decarboxylated intermediates generated by ODCase decarboxylation. The hydrogen exchange rates for H5 and H6 from 1,3-dimethyluracil were recently measured [23]; interestingly, the rate of H5 exchange is accelerated by acetate, and the exchange rate at H5 is somewhat faster than that at H6 with moderate acetate concentration. If some fraction of the
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Fig. 1. Possible reaction mechanisms for decarboxylation of OMP by ODCase addressed in this study. The general acid for protonation of the substrate is likely to be a catalytic lysine side chain present at active sites of all ODCases (for example, Lys93 of the yeast ODCase [2]). (Reaction A) O2 protonation mechanism proposed for ODCase catalysis. (Reaction B) Electrophilic substitution mechanism proposed for ODCase catalysis [1,3,4]. (Reaction C) C5 protonation mechanism proposed for ODCase catalysis [20]. The stereochemistry at C5 indicated has not been proposed and is shown only to emphasize the possible fates of the hydrogens in the reaction. R ¼ ribose-50 -phosphate.
decarboxylated intermediates suggested in the reaction in Fig. 1A undergoes rearrangement to form the 5-anion, resulting from a hydrogen shift from C5 to C6 (Fig. 2), it would account for the Creasey and Handschumacher [22] result and would provide strong evidence for a reaction involving a decarboxylated intermediate capa-
ble of undergoing rearrangement, such as that in the mechanism in Fig. 1A. We noted that 5-fluoroOMP has been shown to be a substrate for ODCase [24] and that a similar fluorine shift from C5 to C6 would be highly unlikely, but we reasoned that the possible hydrogen shift, if it does exist, would necessarily be only partial and not obligatory for the reaction, since Creasey and Handschumacher [22] observed only a partial loss of labeled hydrogen. The proposed electrophilic substitution mechanism (Fig. 1B) does not suggest any possible reaction for H5 that could result in a portion of labeled H5 being lost to solvent or rearranged. Thus, an observed hydrogen shift involving H5 would provide evidence against this direct substitution mechanism. The mechanism shown in Fig. 1C would also be addressed by this study. The decarboxylated product in Fig. 1C must undergo rearrangement to the final product UMP. If this rearrangement were enzymatically facilitated, the proton removed from C5 would likely be the same one that was added to C5 to initiate decarboxylation. Thus, the hydrogen atom present at C5 in OMP would remain in the product. However, if the rearrangement is not enzymatically facilitated, then either hydrogen could be lost to solvent, the amount of hydrogen isotope label present in the product at C5 would be diminished from that in the substrate, and some isotope may even arise at C6. In this study, we prepared [5-2 H]OMP and analyzed the product UMP, decarboxylated by yeast ODCase, for deuterium content at C5 and C6 by 1 H NMR, using the decrease in 1 H signals as an indicator of deuterated substrate or product. Additionally, we examined the possibility that ODCase catalyzes hydrogen exchange in the product UMP, using unlabeled UMP in D2 O with ODCase and determining the 1 H=2 H content by NMR.
Materials and methods [5-2 H ]OMP
Fig. 2. A proposed hydrogen shift in a zwitterionic (A ! B) or anionic (C ! D) decarboxylated intermediate. In both reactions A ! B and C ! D, hydrogen at C5 is proposed to shift to C6. For the nucleotide reaction intermediate, R ¼ ribose-50 -phosphate.
This labeled substrate was prepared according to a previous account [16]. Orotic acid (Sigma Chemical Co.) was added to a mixture of acetyl chloride and D2 O (Cambridge Isotope Laboratories) and heated under reflux for 4 days. The resulting [5-2 H]orotic acid was analyzed for 1 H=2 H content in a sample dissolved in deuterated dimethyl sulfoxide (Cambridge Isotope Laboratories), using a Varian Gemini 2000 400-MHz NMR spectrometer and comparing the integration of the H5 signal to those for the H1 and H3 signals. The deuterated orotate was then converted to OMP using orotate phosphoribosyltransferase (a generous provision from the laboratory of Dr. Charles Grubmeyer, Temple University) and 5-phosphoribosyl-1-pyrophosphate (Sigma Chemical Co.). The resulting OMP was purified
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Fig. 3. (A) Portions of 1 H NMR spectrum of UMP in D2 O. H6 signal (left, 7.9–8.0 ppm); H5 and H10 signals overlapping (right, 5.8 ppm). (B) Portions of 1 H NMR spectrum of UMP in D2 O, same signals as in A, following a 14-h reaction with 100 lg yeast ODCase.
by HPLC using conditions previously described for purification of 2-thioOMP [18]. [5-2 H]OMP was also analyzed for deuterium content by NMR. The [5-2 H]OMP sample was prepared in D2 O, using repeated evaporation and redissolving with D2 O. The integration of the 1 H signal for H5 was compared to that for the H10 signal.2 The sample was then evaporated to dryness and redissolved several times with water to remove exchangeable deuterium. [5-2 H]OMP (1.9 mM) was then decarboxylated in 50 mM Tris, pH 8.0, with yeast ODCase, purified as described previously [25]. The decarboxylated sample was evaporated to dryness and redissolved in D2 O, and the resulting UMP was then analyzed for deuterium content without purification. Assessment of hydrogen exchange in UMP catalyzed by ODCase A solution of 13.6 mM UMP (Sigma Chemical Co.) was prepared in D2 O with 10 mM Tris buffer, pH 8.0, and analyzed for deuterium content by NMR (H6, doublet, 7.9–8.0 ppm; H5, doublet, 5.8 ppm; H10 , doublet, 5.8 ppm). ODCase (100 lg) was added, and this reaction mixture was incubated at room temperature for 14 h. The UMP was analyzed for deuterium content in this reaction mixture without purification. Results and discussion Analysis of 1 H=2 H content of UMP under our conditions is complicated by the overlap of 1 H NMR signals from H5 and H10 ; both protons yield signals at 2 In this analysis, we assume that H10 of OMP and UMP are not exchangeable either in solution or at the ODCase active site.
5.8 ppm. The upfield half of the H10 doublet is almost exactly coincident with the downfield half of the H5 doublet. Nonetheless, the 1 H=2 H content of H5 and H6 of UMP produced from ODCase decarboxylation of [5-2 H]OMP can be assessed as a ratio of integrations of the H6 signal and the combined signals of H5 and H10 . ODCase does not catalyze hydrogen exchange of UMP to an extent detectable under our conditions. Fig. 3A shows the 1 H NMR spectrum of signals from UMP H6, H5, and H10 before addition of ODCase; this pattern is unchanged by the addition of ODCase (Fig. 3B). The integral ratio of 1:1.8 [for H6 to (H5 + H10 )] is slightly lower than the expected ratio of 1:2, but the ratio does not change during the course of the attempted reaction. No detectable presence of singlets for the H5 or H6 signals, which would indicate deuterium substitution at the neighboring position, is apparent. This result is necessary to examine, without complications, any possible hydrogen shift occurring in an ODCase decarboxylated intermediate. The deuterated orotate and OMP were found by NMR analysis to be only 72% deuterated (Fig. 4A; data not shown for orotate). Partially deuterated OMP can be used for analysis of a possible hydrogen shift from C5 to C6 in the ODCase reaction. The measured integral ratio in the UMP product, H6/(H6 + H5 + H10 ), will be a function of the fraction of 1 H in the partially deuterated OMP (fH ) and the degree of hydrogen shift in the ODCase reaction, expressed as fraction of retention fR (where fR ¼ 1:0 indicates no hydrogen shift and fR ¼ 0 indicates 100% hydrogen shift), according to the following equation: Integral ratio ¼ ffR þ ½ð1 fR Þ fH g=ð2 þ fH Þ:
ð1Þ
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Fig. 4. (A) Portions of 1 H NMR spectrum of partially deuterated OMP. 1 H signal for H5 (left, 5.53 ppm) is decreased compared to 1 H signal for H10 (right, 5.27 ppm) due to partial deuteration at C5. Integration of these signals indicates 28% 1 H at C5, or 72% deuteration, compared to H10 . (B) Portions of 1 H NMR spectrum of partially deuterated UMP, resulting from decarboxylation of partially deuterated OMP by ODCase. 1 H signal for H6 (left, 7.9 ppm) is a mixture of a doublet (for 1 H5 UMP) and a singlet (for 2 H5 UMP) centered between the two halves of the doublet. 1 H signal for H5 and H10 are overlapping (right, 5.8 ppm); the upfield half of the larger H10 doublet is coincident with the downfield half of the smaller H5 doublet. The integrations of these mixed signals are related to the extent of hydrogen exchange from C5, as discussed in the text.
This equation describes the integral ratio H6/(H6 + H5 + H10 ) because the first fR in the equation describes the relative amount of 1 H arising at H6 from solvent (i.e., if fR ¼ 1:0, then all H6 comes from 1 H2 O solvent), and [ð1 fR Þ fH ] describes the relative amount of 1 H arising at H6 due to transfer from H5. The plots of integral ratio versus fR are linear because Eq. (1) can be rearranged to the following equation: Integral ratio ¼ ½ð1 fH Þ=ð2 þ fH Þ fR þ fH =ð2 þ fH Þ: ð2Þ Fig. 5 illustrates the relationship between a measured UMP 1 H NMR integral ratio H6/(H6 + H5 + H10 ) and the degree of hydrogen shift (expressed as fR ) that must occur to yield the measured UMP integral ratio, when the OMP used in the decarboxylation reaction is deuterated to different extents. The steeper slope is for 100% deuterated OMP (% 1 H5 ¼ fH ¼ 0), which would be most sensitive for detection of a small fraction of hydrogen shift. The plot with intermediate slope is for our 72% deuterated OMP (fH ¼ 0:28) and was considered to be sufficiently sensitive for these trials. We measured the integral ratio H6/(H6 + H5 + H10 ) of UMP produced from ODCase decarboxylation of 72% deuterated OMP and used Fig. 5 to determine the fraction of hydrogen retention at C5. Fig. 4B shows the 1 H NMR spectrum of UMP produced from ODCase decarboxylation of partially deuterated OMP. Two features of this spectrum show that hydrogen at C5 is completely retained at C5 in the decarboxylated product, within the limits of detection. First, the integral ratio H6/(H6 + H5 + H10 ) is 0.44; from
Fig. 5. Graph showing relationship of 1 H NMR integral ratios of the signals corresponding to H6, H5, and H10 in partially deuterated UMP resulting from the decarboxylation of partially deuterated OMP and the degree of hydrogen retention that must be present in the ODCase reaction to yield the respective integral ratios. The solid lines represent the linear relationships between the integral ratios and the degree of hydrogen retention for OMP with varying degrees of deuteration. The dashed line represents the measured ratio of integrals, H6/ (H6 + H5 + H10 ), of 0.44, which intersects with the plot for 72% deuterated OMP at 100% hydrogen retention.
Fig. 5, using the plot for 72% deuterated OMP, this value correlates with 100% hydrogen retention at C5. Second, the signal for H5, although partially shrouded by the signal for H10 , is entirely a doublet. If some deuterium had shifted from C5 to C6 during the decarboxylation reaction, some of the H5 signal would be a singlet (representing 1 H5-2 H6-OMP) situated between
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the two peaks of the doublet (representing 1 H5-1 H6OMP). We conclude therefore that no detectable hydrogen loss or shift from C5 is occurring in the ODCase decarboxylation of OMP. Was it reasonable to expect that a C5 to C6 hydrogen shift might be occurring? The result from Creasey and Handschumacher [22] seemed interesting, but could be explained by other factors. H5 is observed to exchange faster than H6, albeit with high temperature and acetate required [23]. Since no hydrogen shift is observable, one must conclude that if a zwitterionic decarboxylated intermediate such as that shown in Fig. 1A does exist in the ODCase reaction, it must be incapable of rearranging. Unfortunately, the results obtained in this study provide little distinction between the proposed ODCase mechanisms. The reaction illustrated in Fig. 1B would not be expected to demonstrate a hydrogen shift at all. The reactions in Figs. 1A and C might result in some product with hydrogen rearrangement, as suggested by the original tritium-labeling data, but neither mechanism requires a hydrogen shift and none was found in our deuterium-labeling study. The current study provides evidence against the possibility that a decarboxylated intermediate as shown in Fig. 1C is released from the active site and rearranges to UMP spontaneously; if this was the case, we would expect to find a higher proportion of the amount of doublet in the H6 signal in the decarboxylated product (7.9 ppm, Fig. 4B), representing loss of deuterium from C5 of the decarboxylated product.
Acknowledgments We gratefully acknowledge the provision of orotate phosphoribosyltransferase from Dr. Charles Grubmeyer. This work was supported by NIH Grant GM63504-01, in part by the Youngstown State University Presidential Academic Center for Excellence in Research, Center for Biotechnology, and by Grant
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34650-B4 from the American Chemical Society Petroleum Research Fund.
References [1] T.C. Appleby, C. Kinsland, T.P. Begley, S.E. Ealick, Proc. Natl. Acad. Sci. USA 97 (2000) 2005–2010. [2] B.G. Miller, A.M. Hassell, R. Wolfenden, M.V. Milburn, S.A. Short, Proc. Natl. Acad. Sci. USA 97 (2000) 2011–2016. [3] N. Wu, Y. Mo, J. Gao, E.F. Pai, Proc. Natl. Acad. Sci. USA 97 (2000) 2017–2022. [4] P. Harris, J.C. Navarro-Poulsen, K.F. Jensen, S. Larsen, Biochemistry 39 (2000) 4217–4224. [5] M.A. Rishavy, W.W. Cleland, Biochemistry 39 (2000) 4569–4574. [6] W.Y. Feng, T.J. Austin, F. Chew, S. Gronert, W. Wu, Biochemistry 39 (2000) 1778–1783. [7] A. Warshel, M. Strajbl, J. Villa, J. Florian, Biochemistry 39 (2000) 14728–14738. [8] D.J. Porter, S.A. Short, Biochemistry 39 (2000) 11788–11800. [9] B.G. Miller, M.J. Snider, S.A. Short, R. Wolfenden, Biochemistry 39 (2000) 8113–8118. [10] B.G. Miller, M.J. Snider, R. Wolfenden, S.A. Short, J. Biol. Chem. 276 (2001) 15174–15176. [11] J.K. Lee, K.N. Houk, Science 276 (1997) 942–945. [12] A. Radzicka, R. Wolfenden, Science 267 (1995) 90–93. [13] B.G. Miller, R. Wolfenden, Annu. Rev. Biochem. 71 (2002) 847– 885. [14] P. Beak, B. Siegel, J. Am. Chem. Soc. 98 (1976) 3601–3606. [15] H.L. Levine, R.S. Brody, F.H. Westheimer, Biochemistry 19 (1980) 4993–4999. [16] S.A. Acheson, J.B. Bell, M.E. Jones, R. Wolfenden, Biochemistry 29 (1990) 3198–3202. [17] J.A. Smiley, P. Paneth, M.H. OÕLeary, J.B. Bell, M.E. Jones, Biochemistry 30 (1991) 6216–6223. [18] J.I. Ehrlich, C.-C. Hwang, P.F. Cook, J.S. Blanchard, J. Am. Chem. Soc. 121 (1999) 6966–6967. [19] J.A. Smiley, M.E. Jones, Biochemistry 31 (1992) 12162–12168. [20] J.A. Smiley, K.M. Hay, B.S. Levison, Bioorg. Chem. 29 (2001) 96–106. [21] T.-S. Lee, L.T. Chung, J.D. Chodera, P.A. Kollman, J. Am. Chem. Soc. 123 (2001) 12837–12848. [22] W.A. Creasey, R.E. Handschumacher, J. Biol. Chem. 236 (1961) 2058–2063. [23] A. Sievers, R. Wolfenden, J. Am. Chem. Soc. 124 (2002) 13986– 13987. [24] K. Shostak, M.E. Jones, Biochemistry 31 (1992) 12155–12161. [25] J.A. Smiley, L. Saleh, Bioorg. Chem. 27 (1999) 297–306.
Hydrogen isotope tracing in the reaction of orotidine-50-monophosphate decarboxylase Jeffrey A. Smiley,* Brian J. DelFraino, and Beth A. Simpson Department of Chemistry and Center for Biotechnology, Youngstown State University, One University Plaza, Youngstown, OH 44555, USA Received 19 November 2002, and in revised form 31 January 2003
Abstract The mechanism of the enzyme orotidine-50 -monophosphate decarboxylase (OMP decarboxylase, ODCase) is not fully characterized; some of the proposed mechanisms suggest the possibility of hydrogen rearrangement (shift from C5 to C6 or loss of H5 to solvent) during catalysis. In this study, we sought mechanistic information for the ODCase reaction by examining the extent of hydrogen exchange in the product uridine-50 -monophosphate, in combination with ODCase, at the H5 and H6 positions. In a subsequent experiment, partially deuterated OMP was prepared, and the extent of 2 H5 rearrangement or loss to solvent was examined by integration of 1 H nuclear magnetic resonance signals in the substrate and the resulting enzymatically decarboxylated product. The absence of detectable hydrogen exchange in these experiments limits somewhat the possible mechanisms for ODCase catalysis. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Orotidine-50 -monophosphate decarboxylase; Isotope labeling; Isotope exchange; Decarboxylation; Enzyme mechanism; NMR
Orotidine-50 -monophosphate decarboxylase (OMP decarboxylase, ODCase)1 has been the subject of heightened interest recently ([1–12]; review [13]). The original mechanism proposed for this enzyme reaction [14] involved proton donation to O2, forming a zwitterion intermediate (Fig. 1A). Various mechanistic studies have lent support to this model [15–20]. Four crystal structures of ODCases from different organisms were recently resolved [1–4], showing the C6 portion of the pyrimidine ring of bound ligands, rather than O2, facing the proposed proton-donating lysine side chain. These structures have led to the formulation of an electrophilic substitution mechanism (Fig. 1B) using protonation and direct CO2 elimination. Most recently, another mechanism has been proposed [21] in which C5 is protonated, leading to the formation of a different zwitterion (Fig. 1C). We were intrigued by an experimental detail from a very early report on ODCase [22]. Creasey and Hands* Corresponding author. Fax: 1-330-941-1579. E-mail address: [email protected] (J.A. Smiley). 1 Abbreviations used: OMP decarboxylase, ODCase, orotidine-50 monophosphate decarboxylase.
chumacher [22] prepared [5-3 H]OMP, decarboxylated this labeled substrate with ODCase, and analyzed the resulting tritiated UMP product using a chemical test with Br2 , designed to liberate 3 H specifically from the 5position of the pyrimidine. They found that only 70% of the 3 H label could be liberated from the resulting UMP and attributed this result to an incomplete 3 H release reaction. We have used the bromination of UMP and other uracil analogs in various syntheses and found the reaction to be virtually complete in a short time. Thus, we wondered whether the incomplete 3 H liberation might be a mechanistic clue and whether some of the 3 H originally present at C5 had been transferred to another molecular site. We considered that the proposed O2 protonation mechanism (Fig. 1A) might involve a rearrangement of some fraction of the decarboxylated intermediates generated by ODCase decarboxylation. The hydrogen exchange rates for H5 and H6 from 1,3-dimethyluracil were recently measured [23]; interestingly, the rate of H5 exchange is accelerated by acetate, and the exchange rate at H5 is somewhat faster than that at H6 with moderate acetate concentration. If some fraction of the
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Fig. 1. Possible reaction mechanisms for decarboxylation of OMP by ODCase addressed in this study. The general acid for protonation of the substrate is likely to be a catalytic lysine side chain present at active sites of all ODCases (for example, Lys93 of the yeast ODCase [2]). (Reaction A) O2 protonation mechanism proposed for ODCase catalysis. (Reaction B) Electrophilic substitution mechanism proposed for ODCase catalysis [1,3,4]. (Reaction C) C5 protonation mechanism proposed for ODCase catalysis [20]. The stereochemistry at C5 indicated has not been proposed and is shown only to emphasize the possible fates of the hydrogens in the reaction. R ¼ ribose-50 -phosphate.
decarboxylated intermediates suggested in the reaction in Fig. 1A undergoes rearrangement to form the 5-anion, resulting from a hydrogen shift from C5 to C6 (Fig. 2), it would account for the Creasey and Handschumacher [22] result and would provide strong evidence for a reaction involving a decarboxylated intermediate capa-
ble of undergoing rearrangement, such as that in the mechanism in Fig. 1A. We noted that 5-fluoroOMP has been shown to be a substrate for ODCase [24] and that a similar fluorine shift from C5 to C6 would be highly unlikely, but we reasoned that the possible hydrogen shift, if it does exist, would necessarily be only partial and not obligatory for the reaction, since Creasey and Handschumacher [22] observed only a partial loss of labeled hydrogen. The proposed electrophilic substitution mechanism (Fig. 1B) does not suggest any possible reaction for H5 that could result in a portion of labeled H5 being lost to solvent or rearranged. Thus, an observed hydrogen shift involving H5 would provide evidence against this direct substitution mechanism. The mechanism shown in Fig. 1C would also be addressed by this study. The decarboxylated product in Fig. 1C must undergo rearrangement to the final product UMP. If this rearrangement were enzymatically facilitated, the proton removed from C5 would likely be the same one that was added to C5 to initiate decarboxylation. Thus, the hydrogen atom present at C5 in OMP would remain in the product. However, if the rearrangement is not enzymatically facilitated, then either hydrogen could be lost to solvent, the amount of hydrogen isotope label present in the product at C5 would be diminished from that in the substrate, and some isotope may even arise at C6. In this study, we prepared [5-2 H]OMP and analyzed the product UMP, decarboxylated by yeast ODCase, for deuterium content at C5 and C6 by 1 H NMR, using the decrease in 1 H signals as an indicator of deuterated substrate or product. Additionally, we examined the possibility that ODCase catalyzes hydrogen exchange in the product UMP, using unlabeled UMP in D2 O with ODCase and determining the 1 H=2 H content by NMR.
Materials and methods [5-2 H ]OMP
Fig. 2. A proposed hydrogen shift in a zwitterionic (A ! B) or anionic (C ! D) decarboxylated intermediate. In both reactions A ! B and C ! D, hydrogen at C5 is proposed to shift to C6. For the nucleotide reaction intermediate, R ¼ ribose-50 -phosphate.
This labeled substrate was prepared according to a previous account [16]. Orotic acid (Sigma Chemical Co.) was added to a mixture of acetyl chloride and D2 O (Cambridge Isotope Laboratories) and heated under reflux for 4 days. The resulting [5-2 H]orotic acid was analyzed for 1 H=2 H content in a sample dissolved in deuterated dimethyl sulfoxide (Cambridge Isotope Laboratories), using a Varian Gemini 2000 400-MHz NMR spectrometer and comparing the integration of the H5 signal to those for the H1 and H3 signals. The deuterated orotate was then converted to OMP using orotate phosphoribosyltransferase (a generous provision from the laboratory of Dr. Charles Grubmeyer, Temple University) and 5-phosphoribosyl-1-pyrophosphate (Sigma Chemical Co.). The resulting OMP was purified
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Fig. 3. (A) Portions of 1 H NMR spectrum of UMP in D2 O. H6 signal (left, 7.9–8.0 ppm); H5 and H10 signals overlapping (right, 5.8 ppm). (B) Portions of 1 H NMR spectrum of UMP in D2 O, same signals as in A, following a 14-h reaction with 100 lg yeast ODCase.
by HPLC using conditions previously described for purification of 2-thioOMP [18]. [5-2 H]OMP was also analyzed for deuterium content by NMR. The [5-2 H]OMP sample was prepared in D2 O, using repeated evaporation and redissolving with D2 O. The integration of the 1 H signal for H5 was compared to that for the H10 signal.2 The sample was then evaporated to dryness and redissolved several times with water to remove exchangeable deuterium. [5-2 H]OMP (1.9 mM) was then decarboxylated in 50 mM Tris, pH 8.0, with yeast ODCase, purified as described previously [25]. The decarboxylated sample was evaporated to dryness and redissolved in D2 O, and the resulting UMP was then analyzed for deuterium content without purification. Assessment of hydrogen exchange in UMP catalyzed by ODCase A solution of 13.6 mM UMP (Sigma Chemical Co.) was prepared in D2 O with 10 mM Tris buffer, pH 8.0, and analyzed for deuterium content by NMR (H6, doublet, 7.9–8.0 ppm; H5, doublet, 5.8 ppm; H10 , doublet, 5.8 ppm). ODCase (100 lg) was added, and this reaction mixture was incubated at room temperature for 14 h. The UMP was analyzed for deuterium content in this reaction mixture without purification. Results and discussion Analysis of 1 H=2 H content of UMP under our conditions is complicated by the overlap of 1 H NMR signals from H5 and H10 ; both protons yield signals at 2 In this analysis, we assume that H10 of OMP and UMP are not exchangeable either in solution or at the ODCase active site.
5.8 ppm. The upfield half of the H10 doublet is almost exactly coincident with the downfield half of the H5 doublet. Nonetheless, the 1 H=2 H content of H5 and H6 of UMP produced from ODCase decarboxylation of [5-2 H]OMP can be assessed as a ratio of integrations of the H6 signal and the combined signals of H5 and H10 . ODCase does not catalyze hydrogen exchange of UMP to an extent detectable under our conditions. Fig. 3A shows the 1 H NMR spectrum of signals from UMP H6, H5, and H10 before addition of ODCase; this pattern is unchanged by the addition of ODCase (Fig. 3B). The integral ratio of 1:1.8 [for H6 to (H5 + H10 )] is slightly lower than the expected ratio of 1:2, but the ratio does not change during the course of the attempted reaction. No detectable presence of singlets for the H5 or H6 signals, which would indicate deuterium substitution at the neighboring position, is apparent. This result is necessary to examine, without complications, any possible hydrogen shift occurring in an ODCase decarboxylated intermediate. The deuterated orotate and OMP were found by NMR analysis to be only 72% deuterated (Fig. 4A; data not shown for orotate). Partially deuterated OMP can be used for analysis of a possible hydrogen shift from C5 to C6 in the ODCase reaction. The measured integral ratio in the UMP product, H6/(H6 + H5 + H10 ), will be a function of the fraction of 1 H in the partially deuterated OMP (fH ) and the degree of hydrogen shift in the ODCase reaction, expressed as fraction of retention fR (where fR ¼ 1:0 indicates no hydrogen shift and fR ¼ 0 indicates 100% hydrogen shift), according to the following equation: Integral ratio ¼ ffR þ ½ð1 fR Þ fH g=ð2 þ fH Þ:
ð1Þ
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Fig. 4. (A) Portions of 1 H NMR spectrum of partially deuterated OMP. 1 H signal for H5 (left, 5.53 ppm) is decreased compared to 1 H signal for H10 (right, 5.27 ppm) due to partial deuteration at C5. Integration of these signals indicates 28% 1 H at C5, or 72% deuteration, compared to H10 . (B) Portions of 1 H NMR spectrum of partially deuterated UMP, resulting from decarboxylation of partially deuterated OMP by ODCase. 1 H signal for H6 (left, 7.9 ppm) is a mixture of a doublet (for 1 H5 UMP) and a singlet (for 2 H5 UMP) centered between the two halves of the doublet. 1 H signal for H5 and H10 are overlapping (right, 5.8 ppm); the upfield half of the larger H10 doublet is coincident with the downfield half of the smaller H5 doublet. The integrations of these mixed signals are related to the extent of hydrogen exchange from C5, as discussed in the text.
This equation describes the integral ratio H6/(H6 + H5 + H10 ) because the first fR in the equation describes the relative amount of 1 H arising at H6 from solvent (i.e., if fR ¼ 1:0, then all H6 comes from 1 H2 O solvent), and [ð1 fR Þ fH ] describes the relative amount of 1 H arising at H6 due to transfer from H5. The plots of integral ratio versus fR are linear because Eq. (1) can be rearranged to the following equation: Integral ratio ¼ ½ð1 fH Þ=ð2 þ fH Þ fR þ fH =ð2 þ fH Þ: ð2Þ Fig. 5 illustrates the relationship between a measured UMP 1 H NMR integral ratio H6/(H6 + H5 + H10 ) and the degree of hydrogen shift (expressed as fR ) that must occur to yield the measured UMP integral ratio, when the OMP used in the decarboxylation reaction is deuterated to different extents. The steeper slope is for 100% deuterated OMP (% 1 H5 ¼ fH ¼ 0), which would be most sensitive for detection of a small fraction of hydrogen shift. The plot with intermediate slope is for our 72% deuterated OMP (fH ¼ 0:28) and was considered to be sufficiently sensitive for these trials. We measured the integral ratio H6/(H6 + H5 + H10 ) of UMP produced from ODCase decarboxylation of 72% deuterated OMP and used Fig. 5 to determine the fraction of hydrogen retention at C5. Fig. 4B shows the 1 H NMR spectrum of UMP produced from ODCase decarboxylation of partially deuterated OMP. Two features of this spectrum show that hydrogen at C5 is completely retained at C5 in the decarboxylated product, within the limits of detection. First, the integral ratio H6/(H6 + H5 + H10 ) is 0.44; from
Fig. 5. Graph showing relationship of 1 H NMR integral ratios of the signals corresponding to H6, H5, and H10 in partially deuterated UMP resulting from the decarboxylation of partially deuterated OMP and the degree of hydrogen retention that must be present in the ODCase reaction to yield the respective integral ratios. The solid lines represent the linear relationships between the integral ratios and the degree of hydrogen retention for OMP with varying degrees of deuteration. The dashed line represents the measured ratio of integrals, H6/ (H6 + H5 + H10 ), of 0.44, which intersects with the plot for 72% deuterated OMP at 100% hydrogen retention.
Fig. 5, using the plot for 72% deuterated OMP, this value correlates with 100% hydrogen retention at C5. Second, the signal for H5, although partially shrouded by the signal for H10 , is entirely a doublet. If some deuterium had shifted from C5 to C6 during the decarboxylation reaction, some of the H5 signal would be a singlet (representing 1 H5-2 H6-OMP) situated between
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the two peaks of the doublet (representing 1 H5-1 H6OMP). We conclude therefore that no detectable hydrogen loss or shift from C5 is occurring in the ODCase decarboxylation of OMP. Was it reasonable to expect that a C5 to C6 hydrogen shift might be occurring? The result from Creasey and Handschumacher [22] seemed interesting, but could be explained by other factors. H5 is observed to exchange faster than H6, albeit with high temperature and acetate required [23]. Since no hydrogen shift is observable, one must conclude that if a zwitterionic decarboxylated intermediate such as that shown in Fig. 1A does exist in the ODCase reaction, it must be incapable of rearranging. Unfortunately, the results obtained in this study provide little distinction between the proposed ODCase mechanisms. The reaction illustrated in Fig. 1B would not be expected to demonstrate a hydrogen shift at all. The reactions in Figs. 1A and C might result in some product with hydrogen rearrangement, as suggested by the original tritium-labeling data, but neither mechanism requires a hydrogen shift and none was found in our deuterium-labeling study. The current study provides evidence against the possibility that a decarboxylated intermediate as shown in Fig. 1C is released from the active site and rearranges to UMP spontaneously; if this was the case, we would expect to find a higher proportion of the amount of doublet in the H6 signal in the decarboxylated product (7.9 ppm, Fig. 4B), representing loss of deuterium from C5 of the decarboxylated product.
Acknowledgments We gratefully acknowledge the provision of orotate phosphoribosyltransferase from Dr. Charles Grubmeyer. This work was supported by NIH Grant GM63504-01, in part by the Youngstown State University Presidential Academic Center for Excellence in Research, Center for Biotechnology, and by Grant
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34650-B4 from the American Chemical Society Petroleum Research Fund.
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