896
Biochemistry 1986, 25, 896-904
Iwamori, M., & Nagai, Y. (1979) J. Neurochem. 32,767-777. Iwamori, M., & Nagai, Y. (1981a) J . Biochem. (Tokyo) 89, 1253-1 264. Iwamori, M., & Nagai, Y. (1981b) Biochim. Biophys. Acta 665, 205-213. Iwamori, M., & Nagai, Y. ( 1 9 8 1 ~ )Biochim. Biophys. Acta 665, 2 14-220. Iwamori, M., Sawada, K., Hara, Y., Nishio, M., Fujisawa, T., Imura, H., & Nagai, Y. (1982) J . Biochem. (Tokyo) 91, 1875-1887. Iwamori, M., Shimomura, J., Tsuyuhara, S., & Nagai, Y. (1984) J . Biochem. (Tokyo) 95, 761-770. Kannagi, R., Nudelman, E., & Hakomori, S . (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3470-3474. Kannagi, R., Levery, S., & Hakomori, S . (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2844-2848. Kasai, M., Iwamori, M., Nagai, Y., Okumura, K., & Tada, T. (1980) Eur. J . Immunol. 10, 175-180. Merrick, J. M., Zadarlik, K., & Milgrom, F. (1978) Int. Arch. Allergy Appl. Immunol. 57, 477-480. Miettinen, T., & Takki-Luukainen, 1. T. (1959) Acta Chem. Scand. 13, 856-858. Momoi, T., Wiegandt, H., Arndt, R., & Thiele, H. (1980) J. Immunol. 125, 2496-2500. Muhlradt, P. F., Bethke, U., Monner, D. A,, & Petzoldt, K. (1984) Eur. J . Immunol. 14, 852-858. Nagai, Y., & Iwamori, M. (1980a) ACS Symp. Ser. No. 128, 435-443. Nagai, Y., & Iwamori, M. (1980b) Mol. Cell. Biochem. 29, 81-90. Nagai, Y., Tsuji, S., & Sanai, Y. (1984) in Ganglioside Structure, Function and Biomedical Potential (Ledeen, R.
W., Yu, R. K., Rapport, M. M., & Suzuki, K., Eds.) pp 183-193, Plenum, New York. Nakano, Y., Imai, M., Naiki, M., & Osawa, T. (1980) J . Immunol. 125, 1928-1932. Reichert, R. A., Gallatin, W. M., Butcher, E. C., & Weismann, I. L. (1984) Cell (Cambridge, Mass.) 38, 89-99. Riedl, M., Foster, O., Rumpold, H., & Bernheimer, H. (1982) J . Immunol. 128, 1205-1210. Rosenfelder, G., VanEijk, R. V. W., Monner, D. A., & Muhlradt, P. F. (1978) Eur. J . Biochem. 83, 571-580. Rosenfelder, G., Ziegler, A., Warnet, P., & Braun, D. G. (1982) JNCI, J . Natl. Cancer Inst. 68, 203-209. Ryan, J. L., & Shinitzky, M. (1979) Eur. J . Biochem. 9, 17 1-1 75. Schwarting, G. A., Summers, A., Stout, R. D., Parkinson, D. R., & Waskal, S . D. (1980) Fed. Proc., Fed. Am. SOC.Exp. Biol. 39, 93 1. Sela, B. (1981) Eur. J . Immunol. 11, 347-349. Smith, K. (1984) Immunology Today 5 , 83-84. Svennerholm, L. (1972) Methods Carbohydr. Chem. 4, 464-474. Taki, T., Takagi, K., Kamada, R., Matsumoto, M., & Kojima, K. (1981) J . Biochem. (Tokyo) 90, 1653-1660. Taki, T., Kawamoto, M., Seto, H., Noro, N., Masuda, T., Kannagi, R., & Matsumoto, M. (1983) J . Biochem. (Tokyo) 94, 633-644. Ugorski, M., Nilsson, B., Schroer, K., Cashel, A., & Zopf, D. (1984) J . Biol. Chem. 259, 481-486. Whisler, R. L., & Yates, A. J. (1980) J . Immunol. 125, 2106-21 11. Young, W., Hakomori, S . , Durdik, J., & Henney, C. (1983) J. Immunol. 124. 199-201.
Biosynthesis of Porphyrins and Corrins. 1. ‘H and 13CNMR Spectra of (Hydroxymethy1)bilane and Uroporphyrinogens I and IIIt Jeremy N. S. Evans,$*$Richard C. Davies,$ Alan S. F. Boyd,$ Isao Ichinose,: Neil E. MackenzieJ A. Ian Scott,*J and Robert L. Baxtert Department of Chemistry, University of Edinburgh, Edinburgh EH9 3JJ, U.K., and Center for Biological N M R , Department of Chemistry, Texas A&M University, College Station, Texas 77843 Received June 26, 1985
ABSTRACT: High-field N M R spectroscopic methods have been applied to study the reactions catalyzed by
porphobilinogen (PBG) deaminase and uroporphyrinogen I11 (uro’gen 111) cosynthase, which are the enzymes responsible for the formation of the porphyrin macrocycle. The action of these enzymes in the conversion of PBG, [2,1 1-13C]PBG, and [3,5-13C]PBG to uro’gens I and I11 has been followed by ‘H and 13CN M R , and assignments are presented. The principal intermediate that accumulated was the correspondingly labeled (hydroxymethy1)bilane (HMB), the assignments for which are also presented.
x e biosynthesis of uroporphyrinogen I11 (uro’gen 111,’ 11; Scheme I) from porphobilinogen (PBG, 2) has been the subject of extensive investigation for the past 35 years. Pioneering work by Bogorad and Granick (1953) and Booij and Rimington (1957) has shown that two enzymes are required to ‘Supported by the Science and Engineering Research Council (U.K.) and the National Institutes of Health (AM32034). *University of Edinburgh. 5 Present address: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, M A 02139. l1 Texas A & M University.
0006-2960/86/0425-0896$01.50/0
catalyze the condensation of 4 mol of PBG to give uro’gen 111. These two enzymes are PBG deaminase (EC 4.3.1.8, deaminase) and uro’gen 111cosynthase (EC 4.2.1.75, cosynthase). In the absence of cosynthase, deaminase catalyzes the conversion of PBG into uro’gen I (7)and the latter is not converted into uro’gen I11 by the action of cosynthase. Since the natural Abbreviations: HMB, (hydroxymethy1)bilane; PBG, porphobilinogen; uro’gen 1, uroporphyrinogen I; uro’gen 111, uroporphyrinogen 111; N M R , nuclear magnetic resonance; A L A , 5-aminolevulinic acid; FID, free induction decay.
0 1986 American Chemical Society
IH
AND
13c N M R
VOL. 25, N O . 4, 1986
O F H M B A N D U R O ' G E N S I A N D 111
Scheme I P
o-$02H
ALA dehydratase
NH,
(1).=l2C
+ll b P 2 HzN
H
(2)0 =l2C (a) .=%
(la) O = k
P
P
A
P
A
(3) .=%
(4) 0 =l2C
(%)O = Y
(4s)0 =l3C
D=deaminase C=cosynthetase P
W
A
A
/
\
A
P
(3)0 =12C,R= OH ( 5 a )0 = % , R = OH
P
(2)0 =l2c
(B) 0 =l3C
(8) 0 = 12C,R = NH2 (6a 0) =%, R = NH
OR
A g Y J
p
P
A
(*) .=13C
\
\
A
P
(go=% IC
A
P
-.
(u)o=13c
897
898
BIOCHEMISTRY
isomer uro’gen I11 is formed only with deaminase-cosynthase mixtures, it can be inferred that the true substrate for cosynthase is the immediate product of deaminase or some intermediate between this product and uro’gen I. The structure of the intermediate could possess a rearranged ring D as in uro’gen 111, and this rearrangement could take place at any level between monopyrrole and tetrapyrrole. The role of diand tripyrroles as possible intermediates was assessed [reviewed in Buckley (1977) and Battersby & McDonald (1975)], but anomalous results were obtained. Radmer and Bogorad (1972), and later Davies and Neuberger (1973), presented spectroscopic evidence from trapping experiments in favor of the intermediacy of the tetrapyrrole (aminomethy1)bilane(6), while Battersby et al. (1973a, 1976) showed that the rearrangement of the ring D takes place intramolecularly. Dauner et al. (1976) and later Battersby et al. (1977, 1978a, 1981) confirmed that 6 can undergo an intramolecular rearrangement to give uro’gen 111. On the basis of the intact incorporation of I3C-labeled 6 into uro’gen I11 by the action of deaminasecosynthase, Battersby et al. (1978b) concluded that 6 was the true enzymic intermediate. Subsequently, in experiments carried out in this laboratory, a transient, free intermediate in the reaction of [2,11-I3C]PBG with deaminase was detected by 13CNMR. In a preliminary paper on this work (Burton et al., 1979a), we called this intermediate “pre-uro’gen”and proposed five possible structures (4, 5, 6,9,and lo). Of these, three structures (4,5,and 10) were consistent with the NMR data. Furthermore, pre-uro’gen was shown (Jordan et al., 1979) to act as a substrate for cosynthase in the absence of deaminase, implying that the enzymes act in tandem and not in association as had previously (and has subsequently) been suggested (Bogorad, 1963; Frydman & Feinstein, 1974; Higuchi & Bogorad, 1975; Battersby et al., 1979). Futher work by Battersby et al. (1979) provided evidence that the intermediate was (hydroxymethy1)bilane (HMB, 5). Work in this laboratory confirmed these findings (Scott et al., 1980) but left certain features of the spectra obtained in earlier 13Cand ISN NMR experiments unexplained. Principally, a doublet was observed for the terminal methylene carbon of pre-uro’gen generated from [amino-15N,11-13C]PBG. While it was initially conjectured that this observation was consistent with the intermediacy of the N-alkylated species (4), subsequent model studies have shown that the magnitude of the coupling observed in these experiments is inconsistent with the structure of 4 (Gossauer et al., 1983). In this paper, we describe characterization of the species that accumulate in the reactions catalyzed by the two enzymes deaminase and cosynthase using ‘H and 13CNMR spectroscopy. Implicit in the rationale of this approach is that other possible transient intermediates, such as the azafulvene species (3), the N-alkylated species (4), the uro’gen I tautomer ( 9 ; Scott, 1981), or the spiro intermediate (lo), might be detected in the purified enzyme reaction mixtures by a judicious choice of 13C-enrichedsubstrates. This would allow a distinction to be made between the characteristic resonances of these intermediates (4,9, 10,and 15-17;Scheme 11). EXPERIMENTAL PROCEDURES Materials. All reagents used were of the highest grade obtainable. 5-Aminolevulinic acid (ALA) was purchased from Sigma Chemical Co. and porphobilinogen synthesized by known methods (Furhop & Smith, 1975). [2,1 1-l3C]PBG (81 atom % 13C) and [3,5-13C]PBG (81 atom % 13C) were prepared by the action of partially purified ALA dehydratase from Rhodopseudomonas spheroides (Evans, 1984) on [5-13C]-
EVANS ET AL.
ALA and [4-13C]ALA, respectively. [5-13C]ALA (90 atom % 13C) was synthesized by the method of Battersby et al. (1973b) and [4-13C]-ALA(90 atom % 13C)prepared by the method of Mitta et al. (1 967). All enzyme manipulations were carried out at 4 OC except where stated otherwise. Purification of Deaminase and Cosynthase. Deaminase and cosynthase were purified from R . spheroides (ATCC 17023), which was cultured according to the method of Lascelles (1956). Deaminase was purified by a combination of the methods of Jordan and Shemin (1973) and Davies and Neuberger (1973) described in detail in the accompanying paper (Evans et al., 1986). Cosynthase was purified as follows. R. spheroides cells (200 g of wet cells) were suspended in buffer (400 cm3; 0.5 M potassium phosphate, pH 7.5) and sonicated as for deaminase (see accompanying paper). After centrifugation at 35000g (30 min), the supernatant was decanted off, made 10 mM in /3-mercaptoethanol, and brought to 30% saturation with ammonium sulfate. The mixture was then allowed to stir for 30 min and centrifuged (ZOOOOg, 15 min) and the pellet discarded. The supernatant was brought to 55% saturation with ammonium sulfate, stirred for 30 min, and centrifuged (20000g, 15 min). The pellet was dissolved in a minimum volume of buffer (ca. 50 cm3; 0.01 M potassium phosphate, pH 7.5) and dialyzed overnight against several changes of buffer (0.01 M potassium phosphate, pH 7.5, plus 1 mM p-mercaptoethanol). The dialyzate was then centrifuged at 1OOOOOg-15OOOOg for ca. 5 h, and the brown pellet was discarded. The supernatant was applied to a freshly equilibrated hydroxylapatite column (5 X 10 cm) and eluted with 0.01 M potassium phosphate, pH 6.8 plus 1 mM p-mercaptoethanol at 30 cm3 h-l. The deaminase was retained, whereas cosynthase was not. The pooled cosynthase activity was concentrated (Amicon, PM-10 membrane) to ca. 30 cm3 and applied to a freshly equilibrated DEAE-cellulose column (5 X 12 cm), eluting with a salt gradient of 0.05 M KCl (300 cm3, in 0.01 M potassium phosphate, pH 7.5, plus 2 mM p-mercaptoethanol) to 0.4 M KCl (300 cm3, in 0.01 M potassium phosphate, pH 7.5, plus 2 mM 8-mercaptoethanol) at 30 cm3 h-l. The cosynthase activity eluted just before deaminase. The active cosynthase fractions were pooled, concentrated (Amicon, PM-10 membrane) to ca. 20 cm3, and applied to a freshly equilibrated G-100 Sephadex column, eluting with 0.05 M potassium phosphate, pH 7.5, plus 2 mM /3-mercaptoethanol. The active fractions (ca. 500 units) were then pooled and concentrated (Amicon, PM-10 membrane) to ca. 2 cm3. All columns were monitored for both deaminase activity and cosynthase activity. Deaminase was assayed routinely by consumption of PBG (1 unit of activity is defined as 1 pmol of PBG consumed per hour) according to the method of Bogorad (1972) and cosynthase by the method of Jordan et al. (1980) (1 unit of activity is defined as 1 pmol of uroporphyrin formed per hour). Preparation of HMB and Uro’gens I and III for 13CNMR Studies. Typically, HMB was prepared by dissolving [13C]PBG (5 mg) in buffer (0.4 cm3, 0.05 M sodium pyrophosphate and 90% D 2 0 , pH 8.5, plus 10 pL of 3.0 M NaOH) and incubating this in an NMR tube with purified deaminase (1.6 cm3, 400 units in the same buffer) for 3.5 min at 37 OC, under N 2 in the dark. The reaction was stopped by careful adjustment to pH 12 (25 p L of 3.0 M NaOH) and cooled to 4 “ C for examination. Uro’gens I and I11 were generated as described by Burton et al. (1980). Isomer ratios of uroporphyrins I and I11 methyl
'H AND
13c N M R
O F H M B A N D U R O ' G E N S I A N D 111 P
A(Y-$p \ NH 3
VOL. 2 5 , NO. 4, 1986
A
P
)
P
PEG
6 8
6 2
U
A P
P
HYDROXYMETHYLBILANE
4 0
A
HN /'
HOJ
A
899
URO'GEN I
3.0
2 0 ppm
1: 300-MHz 'H NMR spectra of PBG (2 mg in 0.1 cm3 of 99% D 2 0 adjusted to pH 8 with Na,C03) and deaminase (0.4 cm3, 100 units in 0.05 M potassium phosphate, pH 7.6, buffer) at 4 OC with time (interval between spectra = 199 s). Data were recorded with gated low-power irradiation of the HOD resonance, number of scans (NS) = 48 per FID, relaxation delay = 2 s, acquisition time = 1.64 s, size of data table = 16K, and pulse width = 90°. FIGURE
P
A
A
P
URO'GE N I
A
P
HY D ROXYME THY L B I LANE
300-MHz 'H NMR spectra of PBG (synthetic), HMB (synthetic), and uro'gen I11 (enzymically produced) at pH 8.0 and ,25 OC. Data were accumulated and processed exactly as in Figure 1.
FIGURE 2:
esters were determined by HPLC (Nordlov et al., 1980). NMR Spectroscopy. All NMR spectroscopy was performed on a 7.0497 T Bruker WM-300 wide-bore spectrometer equipped with a Bruker Aspect 2000 computer and a variable-temperature assembly. 'H NMR experiments were conducted at an observation frequency of 300.133 MHz in 5-mm tubes (0.5 cm3) and 13CNMR at 75.473 MHz in 5- (0.5 cm3) or 10-mm tubes (2.0 cm3). RESULTSAND DISCUSSION ' H NMR Studies. In the 'H NMR experiments, unlabeled PBG was incubated with deaminase at pH 8.0 and 4 "C and examined over a time course of 5 h (see Figure 1). Clearly,
the initial resonances of PBG (Evans et al., 1985) decrease in intensity as uro'gen I formation takes place. Additional signals of lower intensity than substrate or product can be seen at 4.4 and 6.4 ppm, corresponding to the HO-CH,-pyrrole and pyrrole+ of HMB (5), respectively. These assignments were confirmed when compared (see Figure 2) with the spectrum of synthetic HMB (A. I. Scott, I. Ichinose, and E. M. Abbott, unpublished work). The absence of signals corresponding to vinyl protons suggests either that the proposed azafulvene species (3) is not an intermediate or that it is not present at detectable concentrations. However, it has been proposed in a recent paper (Falk & Schlederer, 1981) that the exomethylene protons of 3 would
900
E V A N S ET A L .
B IO C H E M IS T R Y
Scheme I1
P
6
b
D=deaminase
C=cosynthetase
HO
NH
chemical
HN
=''C
P
have a chemical shift of ca. 4.9 ppm, which would therefore be obscured by the HOD signal. "C NMR Studies. Uro'gen I (7a) was generated by the action of deaminase on [2,1 1-13C]PBGand examined by 13C NMR at pH 8.0 and 25 OC. The spectrum obtained was essentially the same as that previously described (Burton et al., 1980). The meso carbons occur as a doublet centered on 21.81 ppm ('JCc = 50.3 Hz) and a singlet at 21.81 ppm, corresponding to isolated 13C atoms arising from statistical
A
dilution of I3C (probability of 13C-13C adjacency is 65%). The a-pyrrolic carbons show a similar doublet centered on 124.12 ppm (lJCc = 50.3 Hz). Similarly, uro'gen I11 (lla) prepared from [2,1 1-13C]PBG was analyzed by 13CNMR. The spectrum is shown in Figure 3, which differs from that reported by Burton et al. (1980). The doublet at 20.68 ppm ('.Icc = 50.0 Hz) can be assigned to C-5 and C-10 and the superimposed triplet ('JCc = 50.0 Hz) centered at 20.68 ppm to C-15. A broad singlet due to
'H AND
13c N M R
VOL. 25, N O . 4, 1986
OF HMB AND URO'GENS I AND 111
c
901
P
100
50
20 PPm
3: 'H-decou led (0.8 W continuous) 75.5-MHz 13CNMR spectrum of uro'gen I11 (HPLC analysis: 10% I; 90% 111) derived from incubation of [2,1I-' t3CIPBG (2 mg) in degassed buffer (2.8 cm3,0.025 M potassium phosphate, pH 7.6) with deaminase (0.1 cm3, 10 units) and cosynthase (0.1 cm3, 10 units) in the same buffer at 37 OC for 2 h before lyophilization and dissolved in D20 (99%, 0.5 cm3). The spectrum was recorded at 25 OC,with pulse width = 30°, size of data table = 16K, acquisition time = 0.541 s, and NS = 4000. The FID was zero-filled to 32 K, exponentially multiplied with a broadening of 5 Hz, and Fourier transformed. FIGURE
P
+ Uro' i
A
P
+ Uro' I
__~_ 100
50
20 ppm
'H-decoupled (0.8 W continuous) 75.5-MHz 13CNMR spectrum of HMB derived from incubation of [2,11-13C]PBG( 5 mg in 0.4 cm3 of 0.05 M sodium pyrophosphate and 90% D20, pH 8.5, buffer + 10 WL of 3.0 M NaOH) with deaminase (1.6 cm3,400 units in the same buffer) for 3.5 min at 37 OC, before addition of 3 M NaOH (25 pL) and cooling to 4 OC. Data were accumulated at 4 OC for 36 min with pulse width = 15O, size of data table = 16K, acquisition time = 0.541 s, and NS = 5000. The accumulated FIDs were zero-filled to 32 K, exponentially multiplied with a broadening of 3 Hz, and then Fourier transformed. HPLC analysis of products: 15% I; 85% 111. FIGURE 4:
(2-20 is also observed at 20.68 ppm. The pattern for the pyrrolic carbons (C-4, C-7, C-14, and C-16) is more complex. The doublet centered at 123.47 ppm can be assigned to C-4 and C-9, and the broad doublet at 123.63 ppm can be assigned to C-14 and C-16. In the previous analysis (Burton et al., 1980), the two-bond 13Ccoupling between (2-14 and C-16 was suggested to be of the order of 7.5 Hz. However, the analysis of the 13CNMR spectra of uro'gens I and I11 derived from [3,5-13C]PBGsuggests that the two-bond 13Ccoupling is of the order of 4 Hz (vide infra). Furthermore, the doublet (zJcc = 4 Hz) arising from 13Cat C-14 and C-16 would be expected
to be of low relative intensity, since only 1 1 -6%of the sample would be enriched at C-14 and C-16 but not at C-15. When [2,11-13C]PBG(5 mg) was incubated with deaminase (400 units in sodium pyrophosphate-DzO buffer, pH 8.5) for 3.5 min at 37 OC, adjusted to pH 12, and cooled to 4 OC, the predominant species present was HMB (5a), whose spectrum is shown in Figure 4. All the enriched carbons of HMB can be assigned (Table I), and in addition, residual PBG and uro'gen I are present. In this spectrum, the C-11 and C-2 resonances of residual PBG were observed at 34.87 and 113.48 ppm. At pH 8.0, [2,1 1-13C]PBGexhibits resonances at 34.4
902
BIOCHEMISTRY
E V A N S ET A L .
121.1
126.0
1 17 4
125
117.7
120
/ I
116
FIGURE 5: 'H-decoupled (0.8 W gated) 75.5-MHz "C NMR spectrum of uro'gen I (HPLC analysis: 96% I; 4% 111) derived from incubation of [3,5-"C]PBG (1 mg in 0.25 cm3of 0.1 M sodium pyrophosphate, pH 8.0, buffer) with deaminase (0.05 cm3, 10 units, and 2.2 cm3of 0.025 M potassium phosphate, pH 7.8) for 2 h at 37 OC under N2 in the dark, lyophilization, and dissolution in N,-degassed buffer (0.5 cm3of 0.05 M sodium pyrophosphate and 90% D20, pH 8.0). The spectrum was recorded at 25 OC over a narrow spectral width (1000 Hz) with pulse width = 90°, size of data table = 2K, acquisition time = 1.024 s, and NS = 27 500. The FID was zero-filled to 16 K and Fourier transformed. P
A+& $A -
P
125
120
116 ppm
FIGURE 6: 'H-decoupled (0.8 W gated) 75.5-MHz 13CNMR spectrum of uro'gen 111 (HPLC analysis: 6% I; 94% 111) derived under conditions identical with those of the sample described in Figure 5 with the addition of cosynthase (0.1 cm3, 10 units in the same buffer). Data were accumulated under the same conditions as Figure 5 , NS = 34 250, and the accumulated FIDs were zero-filled to 16 K, exponentially multiplied with a broadening of 1 Hz, and then Fourier transformed.
and 116.1 ppm, which were found to shift to 35.35 and 113.64 ppm at pH 12. Also present in the spectrum of H M B is another peak very close to C-11 of PBG, at 34.68 ppm, which represents a small amount of (aminomethy1)bilane (Burton et al., 1979a; Battersby et al., 1982). The latter arises from trapping of ammonia, produced during the polymerization of PBG, presumably by the azafulvene species (3). To this sample of HMB, purified cosynthase (20 units in phosphate buffer, pH 8.0) was added and the pH adjusted rapidly to 8.0 with HC1. Spectra were recorded at 4 OC every 4.5 min over 2 h, but no new intermediates were detected.
The uro'gens I and I11 generated from [3,5-13C]PBG were characterized at ambient temperature and pH 8.0, over a narrow spectral width (1000 Hz), in order to enhance the resolution. Figure 5 shows the spectrum obtained from uro'gen I, which is a doublet of doublets centered on 117.74 ppm (*Jcc = 4 Hz, 3Jcc = 2 Hz) arising from C-3, C-8, C-13, and C-18 and an identical pattern centered on 126.06 ppm arising from C-1, C-6, C-11, and C-16. The spectrum of uro'gen I11 (see Figure 6) is less well resolved. The doublets at 117.02 (2Jcc = 4 Hz) and 117.34 ppm (2Jcc = 4 Hz) can be assigned to C-13 and C-17, since
AND
1~
13c N M R
VOL. 25, NO. 4, 1986
O F H M B A N D U R O ’ G E N S I A N D 111
903
Y $ J
HO
p
A
130
A
/
PBG P
125
120
ppm
7: ‘H-decoupled (0.8 W continuous) 75.5-MHz I3C NMR spectrum of HMB derived by incubation of [3,5-I3C]PBG (5 mg in 0.4 cm3of 0.05 M sodium pyrophosphate and 80% DzO, pH 8.5) with deaminase (1.6 cm3, 272 units in 0.05 M sodium pyrophosphate and 80% DzO,pH 8.5, buffer) at 37 OC for 4 min before adjustment to pH 12 with 3.0 M NaOH (25 pL) and cooling to 5 OC. Data were accumulated at 5 OC over 36 min with pulse width = 75O, size of data table = 8K, acquisition time = 0.270 s, and NS = 10000. The FID was zero-filled to 32 K, Gaussian multiplied with a Gaussian broadening of 0.350 and a line broadening of -3 Hz, and Fourier transformed. HPLC analysis of products: 21% I; 79% 111. FIGURE
Table I: I3C NMR Assignment of [2,11-I3C]PBG-DerivedHMB
Table 11: I3C NMR Assignment of [3,5-”C]PBG-Derived HMB
(Sa)
(14)
P
A
P
r! ’A *
NH
HN
HO l 2 0
A
6 (PP4”
1 3 c
C-15 c-10
c-5 c-20
C-19
20.7 21.1 21.2
53.8
112.5 C-14 123.1 [d, 1J(13C-13C)= 50 Hz] c-9 123.9 [d, ‘J(I3C-”C) = 50 Hz] 125.7 [d, 1J(’3C-13C)= 50 Hz] c-4 a Indirectly referenced to p-dioxane, 6c = 66.5 ppm.
these carbons are symmetrical in relation to one another and are only coupled to C-11 and C-19, respectively. The unresolved double doublets a t 117.39 and 117.56 ppm can be assigned to C-3 and (2-8, not necessarily respectively. The unresolved double doublets at 125.90 and 125.81 ppm can be similarly assigned to C-6 and (2-11. Finally, the unresolved double doublet at 125.52 ppm can be assigned to C-1, which is in a unique spin system with C-19, with a chemical shift similar to C-1 of uro’gen 1. Similarly, the partially unresolved double doublet at 125.33 ppm (2Jcc = 4 Hz) can be assigned to C-19. In a second experiment under optimized NMR conditions, [3,5-I3C]PBGwas incubated with deaminase a t 37 OC for 4
P
A
P
”C c-8
C-13 c-3 c-18
C-16 C-6 c-11 c-1
6 (PPm)“
116.63 116.75 117.25 121.42
124.99 125.44
125.69 127.28 [d, zJ(13C-’3C)= 4 Hz] = 66.5 ppm.
” Indirectly referenced to p-dioxane, 6,
min, adjusted to p H 12, and cooled to 0-5 OC. A clearly resolved spectrum of H M B was obtained and is shown in Figure 7. Here, all eight carbons are distinguishable, and the assignments are given in Table 11. Also evident in the spectrum are peaks assignable to uro’gen I at 117.54 (C-3, C-8, C-13, C-18) and 125.98 ppm (C-1, C-6, C-11, C-16) and residual [3,5-13C]PBG at 121.42 (C-3 coincident with C-18 of HMB) and 129.16 ppm ((2-5). The latter chemical shift for C-5 of PBG was assigned on the basis of a titration study of PBG (Evans et al., 1985). This sample of H M B was readjusted back to pH 8.0 with HCI after addition of cosynthase (30 units) with rapid mixing, and spectra were accumulated at 4.5-min intervals over 3 h
904 B I O C H E M I S T R Y and at 4 O C . While HMB (5) was consumed with concomitant formation of uro’gen 111, no new sp3 or sp2 carbon-bearing intermediates were detected. ACKNOWLEDGMENTS We thank Stuart Miller for growing the bacteria. Registry No. 2, 487-90-1; 5, 71861-60-4; 7, 1867-62-5; 11, 1976-85-8; C , 9074-91-3; D, 37340-55-9.
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