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Determination of carbonyl oxygen exchange rates in α-ketoacids by gas chromatography-mass spectrometry
ANALYTICAL
BIOCHEMISTRY
Determination
TENKASI Departments
123, 295-302
(1982)
of Carbonyl Oxygen Exchange Rates in cu-Ketoacids Gas Chromatography-mass Spectrometry
S. VISWANATHAN,
CHARLES
E. HIGNITE,*
of Biochemistry and *Pharmacology, University Administration Medical Center, Kansas
AND HARVEY
of Kansas Medical Center, City, Missouri 64128
by
F. FISHER and Veterans
Received December 3 1, I98 1 A method for determining the rate of exchange of the carbonyl oxygen in ~-ketoglutarate using gas chromatography-mass spectrometry is described. The method is based on a new procedure for quenching the exchange reaction by rapid oxidative decarboxylation of the LYketoacid to the next lower homologous carboxylic acid using concentrated hydrogen peroxide. Using this method the rate constant for carbonyl oxygen exchange in ol-ketoglutarate in 0.1 M imidazole buffer, pH 7.5, was found to be (2.8 + 0.2) X lo-’ s-’ at 25’C. Data obtained using this technique suggest that hydration is the mechanism for carbonyl oxygen exchange in ru-ketoacids. The method is also applicable to the measurement of oxygen exchange rates of other Lu-ketoacids free in solution and bound in enzyme complexes.
diol) forms, measurements of the rate of hydration and the equilibrium constant for the keto ti gem-diol equilibrium could be used to estimate their oxygen exchange rates. A variety of techniques including uv spectrophotometry ( 10,l 1 ), NMR spectroscopy using ‘H ( 12) and I70 (13) nuclei, dilatometry ( 14), calorimetry ( 15), polarography (16), pressure-jump (17), and temperature-jump ( 18) relaxation methods have been used to measure the rate of hydration in these compounds. Although most of these techniques give accurate rate constants, we required a method for measuring the oxygen exchange rates of cY-ketoacids in microgram amounts present either free in solution or bound in enzyme complexes. We describe here a new method applicable to ‘*O-enriched a-ketoacids which is based on a chemical procedure for quenching carbony1 oxygen exchange and uses gas chromatography-mass spectrometry (GC-MS)’ for the analysis of “0 content.
Isotopic exchange reactions of oxygen are very useful mechanistic probes (l-5). Most detailed studies of oxygen exchange with water have been done with simple oxygencontaining compounds such as phosphates (6), alcohols, phenols, carboxylic acids, and their derivatives (5). However, few such studies have been done on carbonyl compounds (5). These compounds in general, and a-ketoacids in particular, pose special problems owing to the rapidity of the exchange and the difficulty in separating, drying, and analyzing the products for isotopic oxygen. Direct methods for measuring oxygen exchange rates in “O-enriched compounds using 13C NMR spectroscopy (7) and ir spectroscopy (8) have limited application due to their limited range of time scales, sensitivity, or both. Carbonyl compounds, however, have one feature which permits the indirect estimation of their oxygen exchange rates: they are typically hydrated in aqueous solution (9). On the assumption that the route for oxygen exchange (5, 9) in these compounds is through the formation of hydrated (gem-
’ Abbreviation used: CC-MS, gas chromatographymass spectrometry. 295
0003-2697/82/100295-08$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
296
VISWANATHAN,
MATERIALS
AND METHODS
HIGNITE,
AND
FISHER
solid acid in the case of ‘*O-enriched a-ketoglutaric acid. The solution was incubated for 3 min at room temperature. The excess diazomethane was driven off with a dry nitrogen stream. Prolonged incubation of the solutions with diazomethane leads to the insertion of an additional methylene group at the carbonyl group for a-ketoglutaric acid (20).
Water 97-98% enriched with I80 was obtained from Yeda Research and Development Company, Rehovot, Israel. Bovine liver glutamate dehydrogenase was purchased from Boehringer-Mannheim. cr-Ketoglutaric acid and N-methyl-N-nitroso-ptoluenesulfonamide were obtained from Sigma. Hydrogen peroxide (50%, w/w) and imidazole (99%) were purchased from Fisher Kinetics of the Oxidative Decarboxylation Scientific and Aldrich Chemical Company, of ~-Ketogiutarate respectively. N-Methyl-iV-nitrosourea was obtained from ICN. All the chemicals were The reaction was followed by monitoring used without further purification. the disappearance of the 320-nm cY-ketogluDiazomethane for esterifying **O-labeled tarate absorption at several concentrations or unlabeled succinic acid and unlabeled of HzOz in 0.100 mM imidazole buffer (pH ~-ketoglutaric acid was prepared from N7.55) at 25°C. The concentration of cu-kemethyl-N-nitroso-p-toluenesulfonamide ( 19). toglutaric acid was 5.00 mM and the HZOZ Since the possibility exists that the isotopiconcentration varied from 25 to 200 mM. tally labeled carbonyl oxygen may be lost The concentration of H202 was determined due to hydration or ketal formation, the es- using the absorption coefficient c230= 61 M-’ terification of 2-E2-“01-ketoglutaric acid cm-’ (21). was done using an anhydrous ethereal solution of diazomethane containing no alcohol Kinetics of the Keto S Gem-diol as a precautionary procedure. DiazomethInterconversion in ~-Ketog~utarute ane for this purpose was prepared from The conversion of the gem-diol form to N-methyl-iV-nitrosourea using the same the keto form after a jump in the pH from method (19) but excluding methanol. n-Ketoglutaric acid labeled with “0 at 1 to 7.6 was followed by monitoring the inthe carbonyl group was prepared by incu- crease in the carbonyl absorbance at 250 or bating the acid (251 mg) with 180-enriched 320 nm spectrophotometrically. The de(97.3%) water (0.25 ml) at 23°C for 5 min tailed experimental procedure has been dewith vigorous stirring on a vortex stirrer. The scribed previously ( 11). solution was frozen imm~iately in a methanol-dry ice bath and lyophilized. Mass Procedure for Determining Oxygen spectral analysis of the residue showed that Exchange Rates in a-Ketoacids 63.5% of the or-ketoglutarate was labeled with “0 at the carbonyl group with very Solid cr-ketoglutaric acid (1.15 mg) enriched with 180 (-65%) at the carbonyl slight enrichment at the carboxyl groups. Incubation under the same conditions for 20 group was added with rapid stirring to 2.0 h effected the complete exchange of all five ml of 0.1 M imidazole buffer (pH 7.50) at oxygens in cr-ketoglutaric acid with the ox- 25.1 rf: 0.1 “C in a thermostated vessel. The ygens in the medium. buffer solution also contained 2 eq of imidMethyl esters of succinic and a-ketogluazole-free base to neutralize the two cartaric acids were prepared by adding an ethe- boxylic acid groups of a-ketoglutaric acid. real solution of diazomethane to the acid Aliquots of the solution (50 ~1) were withdissolved in methanol and ether or to the drawn periodically (every 25 to 100 s) and
OXYGEN
EXCHANGE
transferred to test tubes ( 13 X 100 mm) containing 0.1 ml of 50% H202 at 25°C. The solutions were thoroughly mixed on a vortex stirrer and the test tubes were cooled in an ice bath. The excess HtOz was destroyed by adding 3-4 drops of bovine liver catalase (~0.25 mg/ml in 0.1 M imidazole buffer of pH 7.50) dropwise to the test tubes. Cooling during this step is essential since the decomposition of H202 is exothermic and the evolution of oxygen is quite brisk at room temperature. The destruction of H202 was assumed to be complete when an additional drop of catalase solution did not produce effervescence. The solutions in the test tubes were lyophilized. The residue containing the imidazole salt of succinic acid was dissolved in about 1 ml of anhydrous ether and anhydrous methanol and esterified with diazomethane. The excess ether was driven off from the solution in a stream of air or nitrogen gas. A small aliquot (5-25 ~1) of the solution containing 5 pg of dimethyl succinate was injected into the GC port of a GCMS system for determining its ‘*O content. The esterification of the lyophilized material with diazomethane leads to significant amounts of ester only if amine buffers, such as imidazole, Tris, etc., which can potentially donate their proton back to succinate to form succinic acid, are employed. Very low recovery of succinate was observed when phosphate was employed as a buffer at the neutral pH. In this case, the solution was acidified to pH 3.7 + 0.2 with 1 M formic acid after the H202 was destroyed and the resulting succinic acid was extracted with 5 ml of ether. Mass Spectral Analysis All mass spectral determinations were done using a Finnigan Model 3300 gas chromatograph-mass spectrometer equipped with a Finnigan 6000 data system. A general purpose 3% OV- 1 glass column (6 ft X l/8 in.) was used for GC analysis with helium at 30 psi as the carrier gas. The GC injector was
297
IN a-KETOACIDS
maintained at 250°C and the column was maintained at 70-80 and 90-100°C for analyzing dimethyl succinate and dimethyl a-ketoglutarate, respectively. The mass spectrometer was run at an ionizing electron voltage of 70 eV. Ion intensities were recorded at I-s intervals and areas under the curves in the appropriate mass fragmentograms were used for quantitation of ‘*O incorporation. In most cases the mass fragmentograms had flat baselines with a sharp peak, whose Rf corresponded to that of the target compound. Fitting of Mass Spectral Kinetic Rate Equations
Data to
The intensities of the isotope peaks of an ion were normalized by setting their sum to 100. The percentage contribution of each isotope peak to the sum was defined as its “normalized intensity.” Rate constants (k) for oxygen exchange were determined by computerized fitting of the normalized intensity (I) versus time (t) data to the equation, Z = C - Aeek’, 111 where C is the normalized isotope peak at equilibrium - A its initial value. validation
of the quenching
intensity of an (t = co) and C Procedure
The following requirements must be met for the procedure employed for quenching carbonyl oxygen exchange and analyzing the product for isotopic content to be successful: 1. The rate of the quenching reaction must be much faster than the rate of carbonyl oxygen exchange reaction. 2. The product of the quenching reaction must retain the labeled oxygen atom. 3. The product should not exchange this labeled oxygen atom with the solvent during the quenching step and in other successive steps needed to prepare the product for isotopic analysis. The oxidative decarboxylation of cu-ketoglutarate to succinate meets all these requirements as explained below.
298
VISWANATHAN,
HIGNITE,
1. Kinetics of decarboxylation of cx-ket~gIutarate. A preliminary kinetic study of the reaction of hydrogen peroxide with CYketoglutarate has been reported (22). More detailed kinetic (23-25) and mechanistic (26) studies have been done using other cyketoacids such as pyruvic, glyoxylic, and phenylglyoxylic (benzoylformic) acids. In all these studies carbon dioxide and a carboxylic acid have been identified as the only products. For example, benzoic acid is produced from benzoylformic acid. The rate of oxidative decarboxylation of a-ketoglutarate is first order in a-ketoglutarate through greater than 95% completion when hydrogen peroxide is in large excess. Linear plots are observed for the dependence of the pseudofirst-order rate constant for the reaction on hydrogen peroxide concentration. Slight deviation to lower rates, however, is observed at high H202 concentrations. The secondorder rate constant for the disappearance of a-ketoglutarate in 0.1 M imidazole buffer, pH 7.55, is 0.44 + 0.02 M-’ se’ at 25°C. The rate constant at pH 5.75 under similar conditions is 0.12 + 0.01 M-’ s-l. Thus, it appears that the half-life of a-ketoglutarate (IV 1 mM) at high concentrations of hydrogen peroxide (12.5 M) is less than 0.2 s in neutral pH solutions at 25°C. In contrast, a typical half-life for carbonyl oxygen exchange in an a-ketoacid is about 10 min at 25°C as reported for pyruvic acid (26). Thus, the oxidative decarboxylation of an cw-ketoacid is over a thousand times faster than the carbonyl oxygen exchange under these conditions. 2. retention of carbonyl oxygen isotope in the oxidative decarboxylation. Brodskii et al. (26) have studied the mechanism of oxidative decarboxylation of pyruvic acid by HzOz with ‘*O-enriched substrates and water. They have clearly shown that one of the carboxyl oxygens in the product, acetate, is derived from Hz02 using HZ r802, which does not undergo significant oxygen exchange with water. However, the enrichment of “0
AND
FISHER
in the acetate was only 0.73% when pyruvic acid enriched to 10.0% and 0.70% with ‘*O at the carbonyl and carboxyl group was decarboxylated with H2 1602. After correcting for the carbonyl and carboxyl oxygen exchange in pyruvic acid under their experimental conditions they calculated that acetic acid should contain 0.80% “0 in excess of the natural content. The large uncertainty introduced by the oxygen exchange raises doubts about the conclusion that “the carbony1 group is oxidized and is retained in the acetic acid that is formed.” Consequently, we have repeated this experiment under conditions where carbonyl oxygen exchange is minimal. The results of this experiment are described below. cu-Ketoglutaric acid (~50 pg) enriched to about 65% with I80 at the carbonyl oxygen was rapidly dissolved in 0.1 ml of 38% H202 in 25 mM imidazole buffer (pH 7.6). The starting material and the resulting succinate were esterified with diazomethane and their “0 content was analyzed with mass spectrometry. The mass spectra of [2-180]dimethyl a-ketoglutarate and that of dimethyl succinate derived from it by rapid oxidative decarboxylation are shown in Fig. 1. Both dimethyl succinate and dimethyl tr-ketoglutarate have no detectable parent ion peaks in their mass spectrum. Consequently, their “0 content was determined by monitoring the intensity of a major fragment containing the 180 isotope. The major fragments derived from both compounds OCcur at m/z 55,59,87, and 115 in the absence of IsO enrichments (20,27). The “O-enriched material produces additional peaks at m/z 57, 61, 89, and 117 in the mass spectrum as shown in Fig. 1. Of the four major ions (m/z 55, 59, 87, and 115) that contain the labeled atom, the ion at m/z 115 was chosen for quantitative isotope analysis because of its high abundance and the low instrumental background at this mass. The 115 ion is derived from both com-
OXYGEN
EXCHANGE
pounds by the loss of one of the two methoxy groups. The 115 ion derived from dimethyl a-ketoglutarate consists of two fragments, +COCO-CH2-CH2-C02CHj and SCH2CH2COC02CH3 with the former probably in larger abundance. Since both fragments contain the carbonyl oxygen, the “0 content of dimethyl a-ketoglutarate determined using the 115 ion should be identical to that obtained using the molecular ion. In dimethyl succinate, on the other hand, the I80 content determined using the 115 ion, +COCH~-CH2-C02CH3, should be only 75% of the value obtained using the molecular ion. The intensity distribution of ions of m/z 115, 117, and 119 observed in the mass spectra of dimethyl a-ketoglutarate and the product, dimethyl succinate, are 28:68:4 and 44:54:2, respectively. The intensity of the ion of m/z 121 is negligible in both cases. The ions of m/z 119 and 121 result from slight ‘*O enrichment at the carboxy1 groups. If the carbonyl oxygen enrichment is calculated from the relative intensities of only the two ions 115 and 117 by neglecting the intensity of 119 ion the ‘“0 enrichments in dimethyl a-ketoglutarate and dimethyl succinate would be 71 and 75%, respectively, indicating quantitative retention of the carbonyl oxygen in the oxidative decarboxylation reaction. The apparently higher I80 content observed in succinate results from the 2% error in the measurement of I80 content and from the inherent errors involved in the simplified calculation. 3. Loss of label from s~c~i~~~~ in handling. The carboxylic acids formed by the
oxidative decarboxylation of the a-ketoacids also exchange their carboxylate oxygens with the oxygen atoms of water although at a much lower rate than carbonyl oxygen (5). The rate of oxygen exchange varies with the hydrogen ion concentration and the rate (R) of oxygen exchange is
Wf[RCM-U
= koDW1
+ ~~~H+l{H~Ol+ hxiEOH-I,
121
299
IN ar-KETOACIDS
5
FIG. 1. Mass spectra of (a) dimethyl a-ketoglutarate enriched with ‘*O at the carbonyl group and (b) dimethyl succinate produced by rapid oxidative decarboxylation of the ‘*O-enriched a-ketoglutaric acid with 38% H,Oz.
where f is the fraction of the total carboxylic acid present as RC02H; k, is the rate constant for the water catalyzed reaction and ku and kOH are the rate constants for hydrogen ion- and hydroxide ion-catalyzed reactions, respectively. At neutral pH and below, the acid-catalyzed exchange is the dominant mechanism and the rate constant for this step (ku) is 7.7 X 10m6 (28) and 1.0 X lo-’ M-* s-’ (29) for acetic acid at 25°C. Thus, the half-life for carboxyl oxygen exchange at pH 3.7 would be 72 days for acetic acid in water at 25°C. The half-life for oxygen exchange in succinic acid should be of this order. Thus, we have shown that rapid decarboxylation of a-ketoacids with concentrated H202 takes place with complete retention of the carbonyl oxygen atom in a product that can be easily analyzed to obtain quantitative data on the isotopic enrichment of the parent cw-ketoacid. Consequently, this reaction pro-
300
VISWANATHAN,
4oo
I185
180
360 540 SECONDS
360 540 SECONDS
HIGNITE,
~ 720
720
Frc. 2. Kinetics of the exchange of i8G atoms from the carbonyl group of oc-ketoglutarate with the i60 atoms of water. The reaction mixture contained ‘*O-enriched cu-ketoglutarate (3.9 mM) and imidazole (0.108 M) in 2 ml of water at 25.1 “C. The pH of the solution was 7.50. The oxygen exchange reaction was quenched by rapid decar~xylation of ~-ketoglutarate with 12.5 M H&. The amount of unexcbanged “0 was determined by mass spectral analysis of the oxidative decarboxylation product, succinate. The normalized intensities of ions with m/z 1I5 and 87 (I,,s/(Iits + I,,,) and Is&, + Is& respectively) in dimethyl sue&ate are shown in a and b, respectively.
vides a useful technique for quenching carbony1 oxygen exchange in or-ketoacids. RESULTS AND DISCUSSION
a-Ketoglutarate The rate of exchange of oxygen atoms between the carbonyl group of W-labeled ~-ketoglutarate and water was measured at 25°C using the rapid oxidative decarboxylation reaction as the quenching procedure. The time dependence of the intensity of the ion at m/z 115 in dimethyl succinate samples at several intervals is shown in Fig. 2a. The intensity shown is the normalized in-
AND FISHER
tensity of the 115 ion expressed as percentage of the total intensities of 115 and 117 ions. The increase in the intensity of 115 ion results from the irreversible loss of the ‘*U isotope from cy-ketogiutarate to the medium. A least-squares exponential fit of the data to Eq. [1] shown by a solid line in Fig. 2a yields a first-order rate constant of (2.7 2~0.2) X lW3 s-i for the carbonyl oxygen exchange. The kinetic experiments are quite reproducible and other trials give first-order rate constants of (2.8 + 0.4) and (2.8 + 0.2) x lo-3 s-‘. The rate of oxygen exchange may also be followed by observing the decrease in the intensity of other fragments which contain the exchangeable oxygen atom such as those at m/z 61 and 83. The intensity of the 87 ion in dimethyl succinate as a function of time is shown in Fig. 2b. The intensity is expressed as percentage of the total intensities of 87 and 89 ions, and the increase in the intensity of the 87 ion results from the loss of the *80 isotope. An exponential fit of the data gave a rate constant of (2.6 +- 0.3) X 10P3 s-’ for oxygen exchange and other trials gave values of (3.0 +- 0.4) and (2.7 & 0.4) X 1OS3s-l. The average rate constant for the carbonyl oxygen exchange in ar-ketoglutarate (1.31 mM) at 25.1 rt O.l”C in 0.108 M imidazoIe buffer (pH 7.50 f 0.03) was found to be (2.8 2 0.2) X f0-3 s-‘. A similar value was also observed in 0.1 M phosphate buffer at the same pH and temperature. The results shown in Figs. 2a and b demonstrate that the exchange of “0 atom from the carbonyl group of ff-ketog~utarate follows first-order kinetics. The precision in the
FIG. 3. Exchange of the carbonyl oxygen “0 isotope of cu-ketoacids with the oxygen atoms of water during the hydration reaction of a-ketoacids.
OXYGEN
EXCHANGE
measurement of the first-order rate constants for oxygen exchange with this technique is comparable to the precision reported for such studies done using “0 nuclear magnetic resonance spectroscopy (30-32). Both techniques yield data which are superior to those reported in the literature (8,26,33). Co~~ar~~o~ of %I Exchange Rates with that Predicted by Hydration ~ea~~re~e~ts Since it appears that the carbonyl oxygen exchange proceeds via a gem-diol intermediate (5,9) (Fig. 3) the first-order rate constant for oxygen exchange should correspond to the rate constant for the slow step of the hydration reaction after statistical corrections. The kinetics of hydration of cY-ketoglutaric acid has been well studied (11) spectrophotometri~~lly, This method measures the rate of approach of gem-diol and ketoform ~ORcentrations to equilibrium levels and gives an overall rate constant k = kh -t- kd, where kd and kh are first-order rate constants for hydrate dissociation and hydrate formation, respectively (Fig. 3). The two rate constants are of course related by k*/k~ = K, the equilibrium constant for the formation of gem-dioi form. Nuclear magnetic resonance spectroscopic studies ( 11,34) of the equilibrium have shown that at 25°C and neutral pH, 7 t 2% of the a-ketoglutarate is present as hydrate with the remainder present as the keto form, The rate constant k for hydration measured by us under conditions identical to those used for measuring K and the oxygen exchange rates is 0.109 -t 0.002 s-‘. The values of kd and kk calculated from k and K are 0. IO2 f 0.003 S-’ and (7 Z?I2) X 1W3 s-j, respectively. The rate constant for the slow step (kh) in the hydration reaction after multiplication by 0.5 to account for the equal probability of losing either the I80 or I60 atom in the dehydration step,z is (3 t 1) X IOW3 s-l which 2 Heavy atom isotope effects on the rate constant for the dehydration step {see Fig. 3) are negligible and kd and k& should be nearly equal.
301
IN u-KETOACIDS
agrees well’ with the observed value for oxygen exchange. Thus, the results are consistent with the proposed mechanism for carbony1 oxygen exchange (5,9).
Although we have presented here results only on the oxygen exchange of cu-ketoglutarate free in solution, the method should be easily extendabfe with minor m~i~cations to acids homologous with cr-ketoglutaric acid and to other cr-ketoacids such as pyruvic acid, oxalacetic acid, and benzoylformic acid. The rates of oxidative decarboxylation of these acids seem to be comparable. For instance, the rate constants for cu-ketogtutarate (pH 7.55, 25°C) and benzoylformic acid (pH 8, 32°C) are 0.44 and 0.63 M-’ s-l (24), respectively. The mass spectra of the methyl esters of the products, acids contain an intense ion, R--C’=0 which facilitates the mass spectral determination of “0 content. Our preliminary experiments reveal that the method is also applicable to studying the earbonyl oxygen exchange rates of tbese com~unds bound to enzyme. For example, we have measured the rate of carbonyl oxygen exchange in ar-ketoglutarate present as a ternary complex with glutamate dehydrogenase and NADP or NADPH. Under the strong oxidizing conditions employed for quenching oxygen exchange, denaturation of the enzyme is quite common. However, this often results in the release of the bound ligands and facilitates their isolation. The experiment required only 400 fig of 180-enriched ff-ketoglutarate and yielded a precise rate constant for the exchange of carbonyl oxygen from a-ketoglutarate bound in the enzyme complex. Thus, the technique appears to be a promising tooj for obtaining ’ Atthoxagh
the agreement
is good
and expected based
on the proposed mechanism, this study, to the best of our knowledge, is the first such study to measure kd, khr and the rate constant for oxygen exchange in the same compound.
302
VISWANATHAN,
mechanistic biochemical
information on chemical reactions of a-ketoacids.
HIGNITE,
and
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BIOCHEMISTRY
Determination
TENKASI Departments
123, 295-302
(1982)
of Carbonyl Oxygen Exchange Rates in cu-Ketoacids Gas Chromatography-mass Spectrometry
S. VISWANATHAN,
CHARLES
E. HIGNITE,*
of Biochemistry and *Pharmacology, University Administration Medical Center, Kansas
AND HARVEY
of Kansas Medical Center, City, Missouri 64128
by
F. FISHER and Veterans
Received December 3 1, I98 1 A method for determining the rate of exchange of the carbonyl oxygen in ~-ketoglutarate using gas chromatography-mass spectrometry is described. The method is based on a new procedure for quenching the exchange reaction by rapid oxidative decarboxylation of the LYketoacid to the next lower homologous carboxylic acid using concentrated hydrogen peroxide. Using this method the rate constant for carbonyl oxygen exchange in ol-ketoglutarate in 0.1 M imidazole buffer, pH 7.5, was found to be (2.8 + 0.2) X lo-’ s-’ at 25’C. Data obtained using this technique suggest that hydration is the mechanism for carbonyl oxygen exchange in ru-ketoacids. The method is also applicable to the measurement of oxygen exchange rates of other Lu-ketoacids free in solution and bound in enzyme complexes.
diol) forms, measurements of the rate of hydration and the equilibrium constant for the keto ti gem-diol equilibrium could be used to estimate their oxygen exchange rates. A variety of techniques including uv spectrophotometry ( 10,l 1 ), NMR spectroscopy using ‘H ( 12) and I70 (13) nuclei, dilatometry ( 14), calorimetry ( 15), polarography (16), pressure-jump (17), and temperature-jump ( 18) relaxation methods have been used to measure the rate of hydration in these compounds. Although most of these techniques give accurate rate constants, we required a method for measuring the oxygen exchange rates of cY-ketoacids in microgram amounts present either free in solution or bound in enzyme complexes. We describe here a new method applicable to ‘*O-enriched a-ketoacids which is based on a chemical procedure for quenching carbony1 oxygen exchange and uses gas chromatography-mass spectrometry (GC-MS)’ for the analysis of “0 content.
Isotopic exchange reactions of oxygen are very useful mechanistic probes (l-5). Most detailed studies of oxygen exchange with water have been done with simple oxygencontaining compounds such as phosphates (6), alcohols, phenols, carboxylic acids, and their derivatives (5). However, few such studies have been done on carbonyl compounds (5). These compounds in general, and a-ketoacids in particular, pose special problems owing to the rapidity of the exchange and the difficulty in separating, drying, and analyzing the products for isotopic oxygen. Direct methods for measuring oxygen exchange rates in “O-enriched compounds using 13C NMR spectroscopy (7) and ir spectroscopy (8) have limited application due to their limited range of time scales, sensitivity, or both. Carbonyl compounds, however, have one feature which permits the indirect estimation of their oxygen exchange rates: they are typically hydrated in aqueous solution (9). On the assumption that the route for oxygen exchange (5, 9) in these compounds is through the formation of hydrated (gem-
’ Abbreviation used: CC-MS, gas chromatographymass spectrometry. 295
0003-2697/82/100295-08$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
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MATERIALS
AND METHODS
HIGNITE,
AND
FISHER
solid acid in the case of ‘*O-enriched a-ketoglutaric acid. The solution was incubated for 3 min at room temperature. The excess diazomethane was driven off with a dry nitrogen stream. Prolonged incubation of the solutions with diazomethane leads to the insertion of an additional methylene group at the carbonyl group for a-ketoglutaric acid (20).
Water 97-98% enriched with I80 was obtained from Yeda Research and Development Company, Rehovot, Israel. Bovine liver glutamate dehydrogenase was purchased from Boehringer-Mannheim. cr-Ketoglutaric acid and N-methyl-N-nitroso-ptoluenesulfonamide were obtained from Sigma. Hydrogen peroxide (50%, w/w) and imidazole (99%) were purchased from Fisher Kinetics of the Oxidative Decarboxylation Scientific and Aldrich Chemical Company, of ~-Ketogiutarate respectively. N-Methyl-iV-nitrosourea was obtained from ICN. All the chemicals were The reaction was followed by monitoring used without further purification. the disappearance of the 320-nm cY-ketogluDiazomethane for esterifying **O-labeled tarate absorption at several concentrations or unlabeled succinic acid and unlabeled of HzOz in 0.100 mM imidazole buffer (pH ~-ketoglutaric acid was prepared from N7.55) at 25°C. The concentration of cu-kemethyl-N-nitroso-p-toluenesulfonamide ( 19). toglutaric acid was 5.00 mM and the HZOZ Since the possibility exists that the isotopiconcentration varied from 25 to 200 mM. tally labeled carbonyl oxygen may be lost The concentration of H202 was determined due to hydration or ketal formation, the es- using the absorption coefficient c230= 61 M-’ terification of 2-E2-“01-ketoglutaric acid cm-’ (21). was done using an anhydrous ethereal solution of diazomethane containing no alcohol Kinetics of the Keto S Gem-diol as a precautionary procedure. DiazomethInterconversion in ~-Ketog~utarute ane for this purpose was prepared from The conversion of the gem-diol form to N-methyl-iV-nitrosourea using the same the keto form after a jump in the pH from method (19) but excluding methanol. n-Ketoglutaric acid labeled with “0 at 1 to 7.6 was followed by monitoring the inthe carbonyl group was prepared by incu- crease in the carbonyl absorbance at 250 or bating the acid (251 mg) with 180-enriched 320 nm spectrophotometrically. The de(97.3%) water (0.25 ml) at 23°C for 5 min tailed experimental procedure has been dewith vigorous stirring on a vortex stirrer. The scribed previously ( 11). solution was frozen imm~iately in a methanol-dry ice bath and lyophilized. Mass Procedure for Determining Oxygen spectral analysis of the residue showed that Exchange Rates in a-Ketoacids 63.5% of the or-ketoglutarate was labeled with “0 at the carbonyl group with very Solid cr-ketoglutaric acid (1.15 mg) enriched with 180 (-65%) at the carbonyl slight enrichment at the carboxyl groups. Incubation under the same conditions for 20 group was added with rapid stirring to 2.0 h effected the complete exchange of all five ml of 0.1 M imidazole buffer (pH 7.50) at oxygens in cr-ketoglutaric acid with the ox- 25.1 rf: 0.1 “C in a thermostated vessel. The ygens in the medium. buffer solution also contained 2 eq of imidMethyl esters of succinic and a-ketogluazole-free base to neutralize the two cartaric acids were prepared by adding an ethe- boxylic acid groups of a-ketoglutaric acid. real solution of diazomethane to the acid Aliquots of the solution (50 ~1) were withdissolved in methanol and ether or to the drawn periodically (every 25 to 100 s) and
OXYGEN
EXCHANGE
transferred to test tubes ( 13 X 100 mm) containing 0.1 ml of 50% H202 at 25°C. The solutions were thoroughly mixed on a vortex stirrer and the test tubes were cooled in an ice bath. The excess HtOz was destroyed by adding 3-4 drops of bovine liver catalase (~0.25 mg/ml in 0.1 M imidazole buffer of pH 7.50) dropwise to the test tubes. Cooling during this step is essential since the decomposition of H202 is exothermic and the evolution of oxygen is quite brisk at room temperature. The destruction of H202 was assumed to be complete when an additional drop of catalase solution did not produce effervescence. The solutions in the test tubes were lyophilized. The residue containing the imidazole salt of succinic acid was dissolved in about 1 ml of anhydrous ether and anhydrous methanol and esterified with diazomethane. The excess ether was driven off from the solution in a stream of air or nitrogen gas. A small aliquot (5-25 ~1) of the solution containing 5 pg of dimethyl succinate was injected into the GC port of a GCMS system for determining its ‘*O content. The esterification of the lyophilized material with diazomethane leads to significant amounts of ester only if amine buffers, such as imidazole, Tris, etc., which can potentially donate their proton back to succinate to form succinic acid, are employed. Very low recovery of succinate was observed when phosphate was employed as a buffer at the neutral pH. In this case, the solution was acidified to pH 3.7 + 0.2 with 1 M formic acid after the H202 was destroyed and the resulting succinic acid was extracted with 5 ml of ether. Mass Spectral Analysis All mass spectral determinations were done using a Finnigan Model 3300 gas chromatograph-mass spectrometer equipped with a Finnigan 6000 data system. A general purpose 3% OV- 1 glass column (6 ft X l/8 in.) was used for GC analysis with helium at 30 psi as the carrier gas. The GC injector was
297
IN a-KETOACIDS
maintained at 250°C and the column was maintained at 70-80 and 90-100°C for analyzing dimethyl succinate and dimethyl a-ketoglutarate, respectively. The mass spectrometer was run at an ionizing electron voltage of 70 eV. Ion intensities were recorded at I-s intervals and areas under the curves in the appropriate mass fragmentograms were used for quantitation of ‘*O incorporation. In most cases the mass fragmentograms had flat baselines with a sharp peak, whose Rf corresponded to that of the target compound. Fitting of Mass Spectral Kinetic Rate Equations
Data to
The intensities of the isotope peaks of an ion were normalized by setting their sum to 100. The percentage contribution of each isotope peak to the sum was defined as its “normalized intensity.” Rate constants (k) for oxygen exchange were determined by computerized fitting of the normalized intensity (I) versus time (t) data to the equation, Z = C - Aeek’, 111 where C is the normalized isotope peak at equilibrium - A its initial value. validation
of the quenching
intensity of an (t = co) and C Procedure
The following requirements must be met for the procedure employed for quenching carbonyl oxygen exchange and analyzing the product for isotopic content to be successful: 1. The rate of the quenching reaction must be much faster than the rate of carbonyl oxygen exchange reaction. 2. The product of the quenching reaction must retain the labeled oxygen atom. 3. The product should not exchange this labeled oxygen atom with the solvent during the quenching step and in other successive steps needed to prepare the product for isotopic analysis. The oxidative decarboxylation of cu-ketoglutarate to succinate meets all these requirements as explained below.
298
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HIGNITE,
1. Kinetics of decarboxylation of cx-ket~gIutarate. A preliminary kinetic study of the reaction of hydrogen peroxide with CYketoglutarate has been reported (22). More detailed kinetic (23-25) and mechanistic (26) studies have been done using other cyketoacids such as pyruvic, glyoxylic, and phenylglyoxylic (benzoylformic) acids. In all these studies carbon dioxide and a carboxylic acid have been identified as the only products. For example, benzoic acid is produced from benzoylformic acid. The rate of oxidative decarboxylation of a-ketoglutarate is first order in a-ketoglutarate through greater than 95% completion when hydrogen peroxide is in large excess. Linear plots are observed for the dependence of the pseudofirst-order rate constant for the reaction on hydrogen peroxide concentration. Slight deviation to lower rates, however, is observed at high H202 concentrations. The secondorder rate constant for the disappearance of a-ketoglutarate in 0.1 M imidazole buffer, pH 7.55, is 0.44 + 0.02 M-’ se’ at 25°C. The rate constant at pH 5.75 under similar conditions is 0.12 + 0.01 M-’ s-l. Thus, it appears that the half-life of a-ketoglutarate (IV 1 mM) at high concentrations of hydrogen peroxide (12.5 M) is less than 0.2 s in neutral pH solutions at 25°C. In contrast, a typical half-life for carbonyl oxygen exchange in an a-ketoacid is about 10 min at 25°C as reported for pyruvic acid (26). Thus, the oxidative decarboxylation of an cw-ketoacid is over a thousand times faster than the carbonyl oxygen exchange under these conditions. 2. retention of carbonyl oxygen isotope in the oxidative decarboxylation. Brodskii et al. (26) have studied the mechanism of oxidative decarboxylation of pyruvic acid by HzOz with ‘*O-enriched substrates and water. They have clearly shown that one of the carboxyl oxygens in the product, acetate, is derived from Hz02 using HZ r802, which does not undergo significant oxygen exchange with water. However, the enrichment of “0
AND
FISHER
in the acetate was only 0.73% when pyruvic acid enriched to 10.0% and 0.70% with ‘*O at the carbonyl and carboxyl group was decarboxylated with H2 1602. After correcting for the carbonyl and carboxyl oxygen exchange in pyruvic acid under their experimental conditions they calculated that acetic acid should contain 0.80% “0 in excess of the natural content. The large uncertainty introduced by the oxygen exchange raises doubts about the conclusion that “the carbony1 group is oxidized and is retained in the acetic acid that is formed.” Consequently, we have repeated this experiment under conditions where carbonyl oxygen exchange is minimal. The results of this experiment are described below. cu-Ketoglutaric acid (~50 pg) enriched to about 65% with I80 at the carbonyl oxygen was rapidly dissolved in 0.1 ml of 38% H202 in 25 mM imidazole buffer (pH 7.6). The starting material and the resulting succinate were esterified with diazomethane and their “0 content was analyzed with mass spectrometry. The mass spectra of [2-180]dimethyl a-ketoglutarate and that of dimethyl succinate derived from it by rapid oxidative decarboxylation are shown in Fig. 1. Both dimethyl succinate and dimethyl tr-ketoglutarate have no detectable parent ion peaks in their mass spectrum. Consequently, their “0 content was determined by monitoring the intensity of a major fragment containing the 180 isotope. The major fragments derived from both compounds OCcur at m/z 55,59,87, and 115 in the absence of IsO enrichments (20,27). The “O-enriched material produces additional peaks at m/z 57, 61, 89, and 117 in the mass spectrum as shown in Fig. 1. Of the four major ions (m/z 55, 59, 87, and 115) that contain the labeled atom, the ion at m/z 115 was chosen for quantitative isotope analysis because of its high abundance and the low instrumental background at this mass. The 115 ion is derived from both com-
OXYGEN
EXCHANGE
pounds by the loss of one of the two methoxy groups. The 115 ion derived from dimethyl a-ketoglutarate consists of two fragments, +COCO-CH2-CH2-C02CHj and SCH2CH2COC02CH3 with the former probably in larger abundance. Since both fragments contain the carbonyl oxygen, the “0 content of dimethyl a-ketoglutarate determined using the 115 ion should be identical to that obtained using the molecular ion. In dimethyl succinate, on the other hand, the I80 content determined using the 115 ion, +COCH~-CH2-C02CH3, should be only 75% of the value obtained using the molecular ion. The intensity distribution of ions of m/z 115, 117, and 119 observed in the mass spectra of dimethyl a-ketoglutarate and the product, dimethyl succinate, are 28:68:4 and 44:54:2, respectively. The intensity of the ion of m/z 121 is negligible in both cases. The ions of m/z 119 and 121 result from slight ‘*O enrichment at the carboxy1 groups. If the carbonyl oxygen enrichment is calculated from the relative intensities of only the two ions 115 and 117 by neglecting the intensity of 119 ion the ‘“0 enrichments in dimethyl a-ketoglutarate and dimethyl succinate would be 71 and 75%, respectively, indicating quantitative retention of the carbonyl oxygen in the oxidative decarboxylation reaction. The apparently higher I80 content observed in succinate results from the 2% error in the measurement of I80 content and from the inherent errors involved in the simplified calculation. 3. Loss of label from s~c~i~~~~ in handling. The carboxylic acids formed by the
oxidative decarboxylation of the a-ketoacids also exchange their carboxylate oxygens with the oxygen atoms of water although at a much lower rate than carbonyl oxygen (5). The rate of oxygen exchange varies with the hydrogen ion concentration and the rate (R) of oxygen exchange is
Wf[RCM-U
= koDW1
+ ~~~H+l{H~Ol+ hxiEOH-I,
121
299
IN ar-KETOACIDS
5
FIG. 1. Mass spectra of (a) dimethyl a-ketoglutarate enriched with ‘*O at the carbonyl group and (b) dimethyl succinate produced by rapid oxidative decarboxylation of the ‘*O-enriched a-ketoglutaric acid with 38% H,Oz.
where f is the fraction of the total carboxylic acid present as RC02H; k, is the rate constant for the water catalyzed reaction and ku and kOH are the rate constants for hydrogen ion- and hydroxide ion-catalyzed reactions, respectively. At neutral pH and below, the acid-catalyzed exchange is the dominant mechanism and the rate constant for this step (ku) is 7.7 X 10m6 (28) and 1.0 X lo-’ M-* s-’ (29) for acetic acid at 25°C. Thus, the half-life for carboxyl oxygen exchange at pH 3.7 would be 72 days for acetic acid in water at 25°C. The half-life for oxygen exchange in succinic acid should be of this order. Thus, we have shown that rapid decarboxylation of a-ketoacids with concentrated H202 takes place with complete retention of the carbonyl oxygen atom in a product that can be easily analyzed to obtain quantitative data on the isotopic enrichment of the parent cw-ketoacid. Consequently, this reaction pro-
300
VISWANATHAN,
4oo
I185
180
360 540 SECONDS
360 540 SECONDS
HIGNITE,
~ 720
720
Frc. 2. Kinetics of the exchange of i8G atoms from the carbonyl group of oc-ketoglutarate with the i60 atoms of water. The reaction mixture contained ‘*O-enriched cu-ketoglutarate (3.9 mM) and imidazole (0.108 M) in 2 ml of water at 25.1 “C. The pH of the solution was 7.50. The oxygen exchange reaction was quenched by rapid decar~xylation of ~-ketoglutarate with 12.5 M H&. The amount of unexcbanged “0 was determined by mass spectral analysis of the oxidative decarboxylation product, succinate. The normalized intensities of ions with m/z 1I5 and 87 (I,,s/(Iits + I,,,) and Is&, + Is& respectively) in dimethyl sue&ate are shown in a and b, respectively.
vides a useful technique for quenching carbony1 oxygen exchange in or-ketoacids. RESULTS AND DISCUSSION
a-Ketoglutarate The rate of exchange of oxygen atoms between the carbonyl group of W-labeled ~-ketoglutarate and water was measured at 25°C using the rapid oxidative decarboxylation reaction as the quenching procedure. The time dependence of the intensity of the ion at m/z 115 in dimethyl succinate samples at several intervals is shown in Fig. 2a. The intensity shown is the normalized in-
AND FISHER
tensity of the 115 ion expressed as percentage of the total intensities of 115 and 117 ions. The increase in the intensity of 115 ion results from the irreversible loss of the ‘*U isotope from cy-ketogiutarate to the medium. A least-squares exponential fit of the data to Eq. [1] shown by a solid line in Fig. 2a yields a first-order rate constant of (2.7 2~0.2) X lW3 s-i for the carbonyl oxygen exchange. The kinetic experiments are quite reproducible and other trials give first-order rate constants of (2.8 + 0.4) and (2.8 + 0.2) x lo-3 s-‘. The rate of oxygen exchange may also be followed by observing the decrease in the intensity of other fragments which contain the exchangeable oxygen atom such as those at m/z 61 and 83. The intensity of the 87 ion in dimethyl succinate as a function of time is shown in Fig. 2b. The intensity is expressed as percentage of the total intensities of 87 and 89 ions, and the increase in the intensity of the 87 ion results from the loss of the *80 isotope. An exponential fit of the data gave a rate constant of (2.6 +- 0.3) X 10P3 s-’ for oxygen exchange and other trials gave values of (3.0 +- 0.4) and (2.7 & 0.4) X 1OS3s-l. The average rate constant for the carbonyl oxygen exchange in ar-ketoglutarate (1.31 mM) at 25.1 rt O.l”C in 0.108 M imidazoIe buffer (pH 7.50 f 0.03) was found to be (2.8 2 0.2) X f0-3 s-‘. A similar value was also observed in 0.1 M phosphate buffer at the same pH and temperature. The results shown in Figs. 2a and b demonstrate that the exchange of “0 atom from the carbonyl group of ff-ketog~utarate follows first-order kinetics. The precision in the
FIG. 3. Exchange of the carbonyl oxygen “0 isotope of cu-ketoacids with the oxygen atoms of water during the hydration reaction of a-ketoacids.
OXYGEN
EXCHANGE
measurement of the first-order rate constants for oxygen exchange with this technique is comparable to the precision reported for such studies done using “0 nuclear magnetic resonance spectroscopy (30-32). Both techniques yield data which are superior to those reported in the literature (8,26,33). Co~~ar~~o~ of %I Exchange Rates with that Predicted by Hydration ~ea~~re~e~ts Since it appears that the carbonyl oxygen exchange proceeds via a gem-diol intermediate (5,9) (Fig. 3) the first-order rate constant for oxygen exchange should correspond to the rate constant for the slow step of the hydration reaction after statistical corrections. The kinetics of hydration of cY-ketoglutaric acid has been well studied (11) spectrophotometri~~lly, This method measures the rate of approach of gem-diol and ketoform ~ORcentrations to equilibrium levels and gives an overall rate constant k = kh -t- kd, where kd and kh are first-order rate constants for hydrate dissociation and hydrate formation, respectively (Fig. 3). The two rate constants are of course related by k*/k~ = K, the equilibrium constant for the formation of gem-dioi form. Nuclear magnetic resonance spectroscopic studies ( 11,34) of the equilibrium have shown that at 25°C and neutral pH, 7 t 2% of the a-ketoglutarate is present as hydrate with the remainder present as the keto form, The rate constant k for hydration measured by us under conditions identical to those used for measuring K and the oxygen exchange rates is 0.109 -t 0.002 s-‘. The values of kd and kk calculated from k and K are 0. IO2 f 0.003 S-’ and (7 Z?I2) X 1W3 s-j, respectively. The rate constant for the slow step (kh) in the hydration reaction after multiplication by 0.5 to account for the equal probability of losing either the I80 or I60 atom in the dehydration step,z is (3 t 1) X IOW3 s-l which 2 Heavy atom isotope effects on the rate constant for the dehydration step {see Fig. 3) are negligible and kd and k& should be nearly equal.
301
IN u-KETOACIDS
agrees well’ with the observed value for oxygen exchange. Thus, the results are consistent with the proposed mechanism for carbony1 oxygen exchange (5,9).
Although we have presented here results only on the oxygen exchange of cu-ketoglutarate free in solution, the method should be easily extendabfe with minor m~i~cations to acids homologous with cr-ketoglutaric acid and to other cr-ketoacids such as pyruvic acid, oxalacetic acid, and benzoylformic acid. The rates of oxidative decarboxylation of these acids seem to be comparable. For instance, the rate constants for cu-ketogtutarate (pH 7.55, 25°C) and benzoylformic acid (pH 8, 32°C) are 0.44 and 0.63 M-’ s-l (24), respectively. The mass spectra of the methyl esters of the products, acids contain an intense ion, R--C’=0 which facilitates the mass spectral determination of “0 content. Our preliminary experiments reveal that the method is also applicable to studying the earbonyl oxygen exchange rates of tbese com~unds bound to enzyme. For example, we have measured the rate of carbonyl oxygen exchange in ar-ketoglutarate present as a ternary complex with glutamate dehydrogenase and NADP or NADPH. Under the strong oxidizing conditions employed for quenching oxygen exchange, denaturation of the enzyme is quite common. However, this often results in the release of the bound ligands and facilitates their isolation. The experiment required only 400 fig of 180-enriched ff-ketoglutarate and yielded a precise rate constant for the exchange of carbonyl oxygen from a-ketoglutarate bound in the enzyme complex. Thus, the technique appears to be a promising tooj for obtaining ’ Atthoxagh
the agreement
is good
and expected based
on the proposed mechanism, this study, to the best of our knowledge, is the first such study to measure kd, khr and the rate constant for oxygen exchange in the same compound.
302
VISWANATHAN,
mechanistic biochemical
information on chemical reactions of a-ketoacids.
HIGNITE,
and
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