Eur. J. Biochem. 65,237-246 (1976)
Enzymic Generation of Chiral Acetates. A Quantitative Evaluation of Their Configurational Assay Helmut LENZ and Hermann EGGERER Fachbereich Biologie und Vorklinische Medizin, Universitit Regensburg (Received December 29, 1975 /February 23, 1976)
1. R-Acetate was generated enzymically from R-acetate in the sequence acetate + malate oxaloacetate acetate, and S-acetate likewise from S-acetate. It was concluded that the formation of malate on malate synthase involves the operation of a normal isotopic effect combined with inversion of configuration. The malate synthase k H / k 2 ” was determined as 3.7 -t 0.5 by a method which yields results independently of the stereochemical purity of the chiral acetates used initially. 2. R-Acetate was also generated from R-acetate in the sequence acetate -+ citrate malate oxaloacetate -+ acetate, and S-acetate likewise from S-acetate. The conclusion is the same as given above, but refers to the formation of citrate on the re-synthase. 3. 2S,3R-[2-2H1,3-2H1,3H1]Malate and 2S,3 S-[2-2H1,3-2H~]malate were prepared from 2 S [2,3-2H3]malate by treatment with fumarase in tritiated water and normal water, respectively. It was assumed that these malate specimens were pure with respect to chirality as generated by isotopic labelling. 4. These two malate specimens were partially converted (about 9%) to acetates in conditions where no racemization at the level of transiently formed oxaloacetate occurred. That no racemization took place was demonstrated experimentally. Oxidative enzymic hydrolysis of 2S,3R[2-2H1,3-2H1,3Hl]malate in normal water and of 2S,3S-[2-2H1,3-2H1]malate in tritiated water produced S-[2H1,3Hl]acetate and R- [’HI ,3Hl]acetate, respectively. 5 . The isolated R-[’HI ,’Hl]acetate and S-[2H1,3H1 ]acetate on configurational analysis yielded malates which in the presence of fumarase retained 79.7 k 0.7% and 20.3 f 0.9%, respectively, of their total tritium content. The symmetric deviation from the 50 2, value found with [3Hl]acetate strenghtens the conclusion that stereochemically pure chiral acetates were analyzed. The malate synthase ~ H / ~ Lwas H determined from the data of this study as 3.9 k 0.2. 6. The average of the values given under paragraphs 1 and 5 for the isotopic discrimination on malate synthase corresponds to k ~ / k = 2~ 3.8 & 0.1. I t was concluded that the configurational analysis of stereochemically pure R-[2H1,3Hl]acetate and S-[’HI ,3Hl]acetate yields malates which in the presence of fumarase retain 79 k 27; and 21 k 2”/;;, respectively, of their total tritium content. Hence, a deviation of 29 f 2 ”/c, from the SO:.;’, value represents the actual amplitude of the configurational assay. 7. Outlines are given for an enzymic generation of chiral acetates in preparative scale. -+
-+
-+
Work performed in cooperation with J. W. Cornforth established a configurational assay of asymmetric methyl groups [2,3]. This was achieved by a combination of purely chemical methods for the synthesis of R-[2H1,3Hl]acetate and S-[’H1~H1]-ace~~
These results are taken from the Thesis of H . Lenz, Universitiit Regensburg 1973; a preliminary account of part of this work has appeared elsewhere [I]. EKJ.~WCS. Alcohol dehydrogenase (yeast) (EC 1 . I . I . 1); citrate (r~)syntliase(EC 4.1.3.28); citrate. (si)synthase (EC 4.1.3.7); fuinarase (EC 4.2.1.2); lactate dehydfogenase (EC 1.1.1.27); malate dehydrogenase (EC 1.1.1.37); inalate synthase (EC 4.1.3.2); oxalacetase (EC 3.7.1.1).
-+
tate, and of purely enzymic methods for their configurational determination in the sequence acetate acetyl-CoA + malate -+ fumarate. R-Acetate and glyoxylate on the enzyme malate synthase produced malate which on incubation with fumarase retained more than 5 0 X , whereas malate derived from Sacetate retained less than 50 ?< of its total tritium content. It was thus possible to determine the absolute configuration of chiral acetates generated in any reaction. Although extensive use of the assay has since been made in the elucidation of the substrate stereochemistry of enzyme-catalyzed reactions, that -+
238
Chiral Acetates
of malate synthase remained ambiguous [3]. The outlined experimental results are consistent with inversion of configuration if a normal isotopic effect, kH > kzH, operates on malate synthase. However, these results are also consistent with retention of configuration if this isotopic effect is inverse, kH < k 2 H ; this is rare, but not unknown [4,5]. The pairs of tritium-labelled malate specimens derived from Racetate on operation of either of the mechanisms are shown, respectively, in Eqns (1) and (2).
+
4
[minor)
[ major I
( minor
1
As can be easily visualized, incubation of each of these pairs with fumarase, which catalyzes an antielimination of the elements of water, must yield identical tritium retentions. The stereochemical course of the malate synthase reaction, therefore, remained to be established (see [3] for full discussion). The same factual situation applies for the recitrate synthase reaction where the elucidated substrate stereochemistry, inversion of configuration during methyl-methylene interconversion, also depends on the operation of a normal isotopic effect [6]. Note that the experimental results are consistent only with the outlined ambiguities; other possibilities such as inversion of configuration combined with the operation of an inverse isotopic effect need not be considered. Another unsettled problem concerned the actual amplitude of the configurational assay. This amplitude represents the difference of tritium retention which is found if malates derived on the synthase from stereochemically pure, e.g. R-acetate, and from symmetrically labelled [3H1]acetate are incubated with fumarase. Malate derived from [3Hl]acetate yields the statistically expected 50 %, that derived from R-acetate yields a higher and that from S-acetate a correspondingly lower retention of tritium [3]. This deviation of tritium from the 50% value is designated as the A 50 value thereafter and depends, of course, on the stereochemical purity of the chiral acetates used. It was found initially that R and S acetates produced malates which on incubation with fumarase retained 69 and 31% (A50% value = 19) of their total tritium content, respectively [3]. The corresponding data were 76 and 25% after
the method of chiral acetate preparation was slightly modified [7a]. Independent work of our Swiss colleagues established a A50% value = 40 [8]. Thus, corresponding the amplitude varies from 19- 40 to apparent intramolecular isotopic effects of k ~ / k 2 ~ (or kzH/kH)= 2.2-9.0. Systematic studies have already shown the reliability of the assay; possible sources of error such as optical instability of the chiral acetates or dependence of the malate synthase k ~ / k 2on~ the concentration of acetyl-CoA, have been excluded [7a]. Furthermore, assuming that a A 50 % value = 40 would represent the true amplitude, it can be calculated that our best samples of chemically synthesized chiral acetates would have been ‘racemic’ to the extent of about 35 %. This however, appeared unlikely. A solution for all these problems is possible in principle, if malates stereospecifically labelled at C-3 are converted into chiral acetates. The enzyme oxalacetase which hydrolytically cleaves oxaloacetate to acetate and oxalate was used for this transformation, firstly because oxaloacetate in the presence of NAD and malate dehydrogenase can easily be generated from malate and secondly because the substrate stereochemistry of oxalacetase is now known [9]. The results are shown in this paper and clarify the outlined ambiguities. An enzymic method for the preparation of chiral acetates is presented additionally.
x,
MATERIALS AND METHODS Enzymes and Suhstrates Enzymes, biochemicals, substrates and methods were as described in the preceding communication [9] with exceptions as follows. The 2S,3R-[3-3H1]malate (98 - 99 % monodeuterated [lo]) used for the generation of chiral acetate in preparative scale, the R- [2H1,3H1]acetate and the S-[2H1,3H~]acetate[7a] (specific activity respectively 7.95 x lo6 and 7.7 x lo6 counts x min-‘ x pmol-’) were a gift from Professor J. W. Cornforth ; re-citrate synthase (specific activity 0.8 U/mg protein) [ l l ] was a gift from Professor G. Gottschalk. Hexadeuteroethanol (>98 % *H) and deuterium oxide (99.75%) were from Merck, Darmstadt. Acetyl-CoA was prepared from R-[2H1,3Hl]acetate by the method of Wieland and and S-t2H1,3H~]acetate Bokelmann and used for enzymic synthesis of 2Rcitrates as described [6] (4.0 pinol acetyl-CoA derived from R-acetate yielded 3.64 pmol 2R-citrate of specific activity 3.68 x lo6 counts 3H x min-’ x pmol-’). The isolated citrate was degraded enzymically to the correspondingly labelled malate (3.5 pmol) as described [6]. The results given here and below for R-acetate were obtained likewise with S-acetate.
H. Lenz and H. Eggerer
All cleavage reactions of stereospecifically labelled malate specimens yielding chiral acetates and oxalate were performed in the presence of 3 mM NAD, 1 mM MnC12, malate dehydrogenase, oxalacetase, lactate dehydrogenase and pyruvate. Cleavage of Malate Formed on Malate Synthase
R-['H1:Hl]Acetate (10 pmol; 7.95 x lo6 counts xmin-') was converted to malate [3] and this was isolated (7.63 pmol; 5.09 x lo6 counts x min-I). [U14 CIMalate (0.21 pmol; 6 x lo5 counts x min-') was added to 2.29 pmol(l.53 x lo6 counts x min-') of the R-acetate-derived malate and the mixture was incubated in a total volume of 0.8 ml with 2.2 U oxalacetase (specific activity 6.65 U/mg protein) and 0.18 U malate dehydrogenase. Pyruvate (0.55 pmol ; slight excess) was added within 25 min and the reaction stopped after 30 min. Unlabelled carrier acetate (2.24 pmol) was added and the product acetate isolated by Bartley diffusion with a yield of 0.58 pmol (57900 counts 3 H ~ m i n - 'and 11300 counts I4Cx min-'; 18% conversion of malate; low yield due to a technical error). This was used for configurational analysis. Cleavage of Malate Derived,from 2R-Citvate
The isolated malate (1.4 pmol; 8.3 x lo5 counts 3H x min-') and 0.11 pmol [U-'4C]malate (5.23 x 10' counts x min-') were incubated in a total volume of 1.05 ml with 0.4 U malate dehydrogenase and 4 U oxalacetase (specific activity 7.5 U/mg protein). Pyruvate (0.36 pmol; slight excess) was added within 25 min and the reaction stopped after 40 min. Unlabelled carrier acetate (2.45 pmol) was added and the product acetate was isolated by Bartley diffusion withayieldof1.75pmol(9.18 x lO4counts3Hxmin-' and 3 . 3 4 lo4 ~ counts I4C xmin-'; 17% inalate conversion). This was used for configurational analysis.
PREPARATION OF STEREOCHEMICALLY PURE CHIRAL ACETATES
239
(0.6 mg protein; 650 U) of a commercial crystal suspension were dissolved and lyophilized twice in 1.0 ml 2H20; the residue was dissolved im 0.4 ml 2H20. 0.1 ml alcohol dehydvogenase (yeast) (3 mg protein, 600 U) of a commercial crystal suspension was dissolved and lyophilized twice in 0.4 ml ' H 2 0 and finally dissolved in 0.4 ml 2H20. The reaction mixture contained deuterated compounds in a total volume of 5.5 mI as follows: Tris buffer, 50 pmol; NAD, 10 pmol; hexadeuteroethanol, 1.75 mmol; alcohol dehydrogenase, 0.25 ml; malate dehydrogenase, 0.25 ml and the rest deuterium oxide; the final pH was 7.5. The total oxaloacetate solution was added in four 0.25-ml samples (about 22 pmol) within about 100 min and the reaction was followed at 366 nm with one fifth of the mixture from the increasing NADH concentration. This was observed after the first two oxaloacetate samples had been added but not thereafter. The reaction was stopped by heat treatment (3 min at 100 "C) after 350 min. [U-14C]Malate (18 nmol; 180000 counts x min-') was added; the mixture was desalted by passage through Dowex-50 (H' ; 1 x 5 cm) and 2S-[2,3-2H3]malate was isolated by chromatography on Dowex-1. The isolated malate (48.8 pmol; 1.76 x lo5 counts x min- ') contained about 0.04% [U-'4C]malate, that is an amount which does not influence the production of stereochemically pure chiral acetates.
2S,3S-[2-2H1,3-2H1]Malate
The reaction mixture in a total volume of 1.0 ml contained 19.5 mM 2S-[2-2H1,3-2Hz]malate,70 mM phosphate buffer, pH 7.5, and 50 U fumarase. The reaction was started with the enzyme and followed at 244 nm (fumarate formation) until the equilibrium between malate and fumarate was established (about 1 min). The mixture was then kept at 40°C for 60 min and the reaction stopped by heat treatment (5 min at 100 "C). 2S,3S-[2-2H1,3-2HI]Malate(12.8 pmol; 66% of theoretical value; about 20% are lost as fumarate) was isolated by chromatography on Dowex-I after the solution had been desalted with Dowex-50 (H').
2S-[2,3-2H3]M~l~tr
The deuteration of starting material was performed as follows. 605 mg Tris were dissolved and lyophilized five times in 1.5 ml deuterium oxide and the residue was dissolved finally in 1.65 ml 2H20 to yield a 3 M solution. 19.9mg NAD (about 25 pmol) were dissolved and lyophilized four times in 1.0 ml 2Hz0; the residue was dissolved in 0.5 ml 'H20. 18.5 mg oxaloacetate (1 10 pmol) were dissolved and lyophilized three times in 1.0 ml 2H20; the residue was dissolved in 1.0 ml 2H20 and neutralized to pH 7.3 with 0.11 ml of the deuterated Tris buffer. 60 pl malate dehydrogenase
2 S,3 R-[2-2H1,3-2H~,3H1]Malate
The reaction mixture in a total volume of 1.0 ml contained : 19.5 mM 2S-[2,3-2H3]malate, 70 mM phosphate buffer, pH 7.5, 0.5 ml tritiated water (0.1 Ci) and 50 U fumarase. The reaction was performed and malate was isolated exactly as described above for 2S,3S-[2-2H~,3-2H~ ]malate but without the addition of [14C]malate. The specific activity of the isolated 2S,3R-[2-3H1,3-2H1,3H1]malate (12.4 pmol; 63.5 y: of theory) was 4.32 x lo5 counts x min-'
Chiral Acetates
240 x pmo1-l and that of tritiated water in the incubation mixture was 9.29 x lo5 counts x min-’ x pmol-I. The results indicate within experimental error that malate had been completely equilibrated with tritiated water.
Generution of S-[2Hl,3H1/ Acetate ,from 2 S,3 R - [ ~ - ’ H I , ~HI - ~,3 H ~ ] M u l u t e The reaction mixture in a total of 1.24 ml contained: 5.58 pmol 2S,3R-[2-2Hl,3-2H~ ,3H1]inalate (about 2.4 x lo6 counts 3H x min-’), 0.54 pmol [U‘“Clmalate (about 2.75 x lo6 counts 14C x min-I), 100 pmol Tris buffer, pH 7.0, 3 pmol NAD, 1 pmol MnC12, 10 U lacetate dehydrogenase, 10.5 U oxalacetase (specific activity 10.7 U/mg protein) and 0.5 U malate dehydrogenase. The latter enzyme was used to start the reaction; the initial rate of malate turnover in these conditions was about 15 of that found when malate dehydrogenase was added in excess. The generated NADH (followed at 366 nm) was oxidized during the reaction by adding 0.16 pmol three times and 0.08 pmol pyruvate once. After a total incubation time of 25 min and just before the reaction was stopped (6 min at 100 “C)the absorbance at 366 nm (NADH) started to increase again. Unlabelled carrier material (2.8 pmol each of acetate and pyruvate; 2.0 pmol each of lactate and malate; 5.0 pmol oxalate) were then added and the mixture was desalted by passage through Dowex-50 (H’ ; 1 x 2.5 cm). Acetate and malate were isolated by chromatography on Dowex-1 (X-8, CI-; 1 x 17 cm) with linear HCI gradients [12]:acids were detected by determining radioactivity and conductivity. Malate was eluted (3.2 ml fractions; flux rate 65 ml/h) with a gradient formed between water (200 ml) and 0.01 N HCI (250 ml) and appeared in fractions 84- 103. Acetate was eluted afterwards with a linear gradient formed between 0.07 N HCI (100 ml) and 0.5 HCI (100 ml) in otherwise unchanged conditions (fractions 45 - 58). The malate-containing fractions were combined and evaporated in vacuo; those containing acetate were treated likewise but neutralized with NaOH before evaporation. The re-isolated 2 S ,3 R- [2-2H ,3-2H ]malate (6.42 pmol; 79 yc,of theory with respect to starting material plus carrier) contained 2.23 x lo6 counts I4C x min-’ and 1.85 x 106counts3Hxmin-’. TheisolatedS-[’HI, 3Hl]-acetate (3.08 pmol; about 2.5 pmol unlabelled carrier material) contained 1.22 x lo5 counts ‘“C x min-’ and 1.45 x lo5 counts 3H x min-’. Generution of R-[’H1 ,3H1]Acetate froin 2S,3S-[2-2H~,3-2H1]M~l~te The partial oxidation and subsequent hydrolysis of 2S,3S-[2-ZH1,3-2Hl]malate(5.61 pmol) and the isolation of acetate and residual malate was performed
exactly as described above for the cleavage of the 2S,3 R-[2-2Hl ,3-2H~ ,3H1]malate specimen but in the absence of [I4C]malate and in the presence of tritiated water (specific activity of 3 H H 0 in the incubation mixture: 9.23 x lo5 counts x min-’ x pmol-’). The re-isolated 2S,3S-[2-2Hl,3-2H~ Jmalate (5.5 pmol) contained 870 counts 3H x min-’ x pmol-’. The isolated R-[2H1,3H1]acetate (3.06 pmol; about 2.5 pmol unlabelled carrier acid) contained 8.87 x lo4 counts x min-‘. OTHER METHODS
The configurational analysis of chiral acetates was performed as described elsewhere [3]. Determination of enzymes, substrates and radioactivity was performed as described in the preceding paper [9]. Acetyl-CoA was determined with citrate synthase [13]. The 3H/’4C ratios given in the tables are the average of several determinations of one and the same sample; the percentage deviations were calculated from these determinations as standard error of the means (SE = s/p). This was also used to calculate ‘average A 50%, values’ from the 3H/’4C ratios given in the tables for the malates derived from R-[2Hl,3H1]acetate and S-[’H1 ,3Hl]acetate (usually six independent determinations). Progress of error for z = x/y was calculated as usual from A ; = xAy/y2 Ax/y.
+
RESULTS AND DISCUSSION INVERSION OF CONFIGURATION O N MALATE SYNTHASE A N D re-CITRATE SYNTHASL
The citrate specimens derived from chiral acetylCoA and oxaloacetate on the re-synthase are labelled stereospecifically at the pro-R side chain and can easily be degraded enzymically to the correspondingly C 3labelled malates [6]. Since the stereochemical ambiguities are the same for both the re-synthase and malate synthase reactions, their solution is reducible to one: that is, how to differentiate between the pairs of malate specimens shown in Eqns 1 and 2. If any specimen of malate containing both [3-2H1?Hl]malate and (33 H ]malate ~ is subjected to oxidative hydrolysis in unlabelled water with malate dehydrogenase and oxalacetase, chiral acetate can only be produced from [3-2H1,3Hl]malate. Since the stereochemistry of the hydrolytic reaction is now known [9], assay of the chirality of this acetate defines the chirality at C-3 of the [3-2H1,3H~]malate. If the 3-methylene group of the malate originated by an enzymic reaction from chiral acetate of known chirality, then the stereochemistry of this reaction is also defined. The absolute magnitude of its kinetic deuterium isotope effect can additionally be deduced from the apparent stereo-
H. Lenz and H. Eggerer
24 1
. malate _-dehydrogenase
___
i
(Ql&2;H +
0
I/
(major) retent ion
t (minor)
(major)
f
--
malate dehydrogenase-oxalacetase
(major)
i
(minor]
Scheme 1
chemical purity of the acetate generated by oxidative hydrolysis. The first malate pair (Eqn 1) will produce R-acetate accompanied with a lesser degree of symmetrically labelled [3Hl]acetate, whereas the second (Eqn 2) will produce S-acetate and a larger amount of [3H1]acetate. This is outlined in Scheme 1. Malare Synthase Chiral acetates, Rand S , were converted enzymically to the malates [3]; one part of the isolated malates was analyzed by incubation with fumarase and another part of each sample was cleaved to acetate and oxalate by oxidative hydrolysis. This was performed by incubation with NAD, oxalacetase, pyruvate, lactate dehydrogenase and limiting amounts of malate dehydrogenase as was described previously [9] and the reactions were stopped after about 20 % substrate had been consumed. The acetates were isolated and again analyzed in the sequence acetate -+ malate + fumarate. The procedure is outlined below for Racetate. R-Acetate(l)+malate(R l)+acetate(Ri)
1 fumarate(R 1)
+ malate(R2)
1 fumarate(R 2)
The experimental results are summarized in Table 1 and show that the R and S acetate (1) specimens on direct analysis yielded malates which retained respectively 76.0 and 24.5% of their total tritium content (average A 50"/, value: 25.81 k 0.24). The acetate(2) specimens, derived from R and S acetate in the sequence acetate(1) -+ malate( 1) + acetate(2), yielded
malates(2) which on incubation with fumarase retained 69.5 and 29.00/,, respectively, of their total tritium content (average A 50% value: 20.24 k 0.37). Thus, R-acetate was generated from R-acetate in the overall reaction, and S-acetate likewise from Sacetate. The results demonstrate that a normal isotopic effect operates during formation of malate on the synthase and that the methyl group is converted into the methylene group with inversion of configuration. A comparison of the A 50 "/, values shows that the stereospecific distribution of tritium at C-3 of the R and S acetate-derived malates(2) corresponds to (20.24 f 0.37)/(25.81 k 0.24) x 100 = 78.5 f 2.2% of that of the malate(1) specimens. The degree of 'racemization' follows as 100-78.5 = 21.5%. This means that on synthesis of the malate(1) specimens, 78.5 of the chiral acetates lost hydrogen and 21.5 2 lost deuterium and corresponds to k H / k 2 H = (78.5 2.2)/(21.3 f 2.2) = 3.7 f 0.5. Note that the intramolecular isotopic effect thus determined is independent of the degree of stereochemical purity of the chiral acetates used initially. re-Citrate Synthase On si-citrate synthase acetyl-CoA adds to the siface of oxaloacetate and the methyl-methylene group transformation proceeds with inversion of configuration [7- 7c]. The stereochemical course of the re-synthase is the opposite in that the acetyl group adds to the re-face of oxaloacetate [14]. It was, therefore, of additional interest to see whether evolution produced an enzyme with completely different stereochemistry, that is whether the condensation reaction
242
Chiral Acetates
Table 1. Loss of tritium from luhelled mulutes, derived,frorn chirul acelutes hcJorr and after the intermediate formation of malate, it1 the presence of fumarase
Table 2. Loss of tritium from labelled malutrs, derived from 2Rcitrutes und f i o m 2 R-ritrutr-derived mulutes, in the presence of famaruse
Malate derivation
Malate derivation via citrate
Time
'H/14C ratios found
average
01 02 03 35 45 60
S-Acetate( 1)
01
02
0.3 35 45 60
Acetate(R2)
01 02 03
5 25 40 60
Acetate(S 2)
01 02
03 5 25 40 60
initial
x
min R-Acetate
c/.
1.801 1.794 1.786 1.376 1.347 1.36X 1.751 1.765 1.753 0.429 0.427 0.435 0.905 0.902 0.901 0.687 0.623 0.627 0.632 0.756 0.760 0.765 0.384 0.222 0.218 0.221
found
100 f 0.2
01 02 03
1.364
27 45 62
76.0 rfr 0.7
Acetate(S 1) 1.765
0.430
0.903
100
k 0.3
*
01 02
03 27 45 62
24.5 rfr 0.6
100
Acetate(R2) 0.2
01 02
03 5
0.627
69.5
i 0.4
0.760
100
0.4
25 40 60
Acetate( S 2)
01
02 03
0.220
'H/14C ratios average
29.0 rfr 0.6
5 25 40 60
cf. initial
x
inin Acetate(f7 1)
1.794
Time
0.342 0.347 0.336 0.225 0.224 0.227 0.434 0.439 0.432 0.146 0.148 0.152 0.656 0.664 0.655 0.537 0.441 0.430 0 439 0.520 0.506 0.520 0.288 0.175 0.164 0.176
0.342
100
k 0.6
0.225
65.8
k 0.4
0.435
100
k 0.4
0.149
34.3 f 0.9
0.658
100 rfr 0.3
0.437
66.4 rfr 0.6
0.515
300 f 0.4
0.172
33.4
k 0.9 ~
above, on incubation with fumarase, yielded tritium retentions of 66.4 and 33.3 respectively (average A 50% value = 16.51 0.40). The results establish that R-acetate was generated from R-acetate in the overall sequence and S-acetate likewise from S-acetate. Thus, the formation of citrate on the re-synthase as well as on the si-synthase involves the operation of a normal isotopic effect and proceeds with inversion of configuration during methyl-methylene group transformation. The chiral acetates used in this study on direct configurational assay produced an average A 50% value of 25.81 f 0.24 (data from Table 1); they yielded citrate derived malates(2) via malates(c) of average Acetate(1) -+ citrate --t malate(c)+acetate(2) + malate(2) A 50"/, value = 16.51 f 0.40. On synthesis of citrate (16.51 0.40)/(25.81 f 0 . 2 4 ) ~100 = 64.0 f 2.1'x 1 1 1 fumarate( 2) of the acetate( 1) specimens therefore lost hydrogen malate(1) + fumarate(1) fumarate(c) and 36.0 & 2.1 lost deuterium (kH/kzH = 1.8 & 0.2). This determination of the intramolecular isotopic The experimental results are summarized in TableZ effect for the re-synthase is again independent of The malates(c) derived from the acetates(1) via the stereochemical purity of the chiral acetates used citrate yielded an average A 50 %,value of 15.86 k0.2 '%;. This provides no new information but is in agreement initially. Other determinations, e.g. from the data for malates(c) in Table 2, which depend on this with previous results [6]. The malate(2) specimens obtained from R and S acetate in the sequence given stereochemical purity are in satisfactory agreement. on the re-synthase proceeded with retention of configuration. Chiral acetates were converted into citrates on the re-synthetase and the isolated citrates were degraded to the malates(c) by incubation with citrate lyase, NADH and malate dehydrogenase [6]. The expected malate specimens are qualitatively those shown in Scheme 1 for their formation from chiral acetyl-CoA and glyoxylate on malate synthase. The malates(c) were subjected to the oxalacetase reaction and the thus-generated acetates(2) were analyz by the configurational assay as usual. The overall procedure consists of the steps shown below.
x)
H. Lenz and H. Eggerer
243
If corrected for the presence of about 10 % ‘racemic’ material in the initial acetates, the data for malates(c) in Table 2 yield kH/kzH= 2.1. It remains to be noted that the A 50% value for the malates(c) would be zero and that for the malate(2) specimens about 14.5 if no isotopic discrimination occurred during formation of citrate on the re-synthase. AMPLITUDE OF THE CONFIGURATIONAL ASSAY
Preparation Stereospecfically Labelled Malates
of
2S,3R- [2-3HL,3-2H1,3Hl]malate and 2S,3S-[22Hl,3-2Hl]nialate were prepared enzymically from oxaloacetate as follows ( c f [lo]). Hexadeuteroethanol was incubated in deuterium oxide with alcohol dehydrogenase and NAD for formation of 4R-[4-’Hl]NADH. Malate dehydrogenase and [3-2H2]oxaloacetate were also present and the generated 4R[4-IH1]NADH was used in situ for formation of 2S-[2,3-’H3]malate with regeneration of NAD. Considerable experimental precaution was taken to ensure a deuterium content of greater than 99% in each of the three labelled positions of the product malate. The isolated trideuterated malate was incubated with fumarase in either tritiated or normal water for formation of 2S,3 R-[2-2H1,3-2H1,3Hl]malate and 2S, 3S-[2-2H1,3-2H1]malaterespectively, and the products were isolated. The procedure is shown in Scheme 2. (alcohol dehydragenasel
LR-IL-~HII NADH
lrnalate dehyjragenase)
\ H02C’
‘H
/C02H
.*‘
2H/ c-c,
’$H
These malate specimens were partially converted (about 9%) to acetates of opposite chirality by incubation in either normal or tritiated water in the presence of NAD with limiting amounts of malate dehydrogenase and excess oxaloacetase. The concentration of oxaloacetate which decreases rapidly in this coupled assay was restored by adding lactate dehydrogenase and titrating NADH with pyruvate as shown below. Malate
Oxaloacetate
NAD
NADH
Lactate
Pyruvate
H20
i i
R
\\
C
(fumarasel
2H
H02C’
C02H
C ‘ =/ H02C/
\2H
I
1_ _ _ _ _ -
[ oxalacetasel------
I C02H
‘C2H2C02H
Acetate + Oxalate
This operation ensured fast turnover of oxaloacetate and thus helped to avoid its racemization. The generated chiral acetates and the residual malates were isolated and their radioactivity determined. The results are sumarized in Table 3 and allow conclusions as follows, In the formation of S-acetate, from the distribution of radioactivity in the isolated acetate, the conversion of [14C]malate can be calculated as at least 8.9%, and that of [3H]malate as at least 6.7%. Thus, [U-14C]malate containing hydrogen at C-2 reacts about 1.33 times faster than 2S,3R-[2-2H1,3-2H1?H~]malate containing deuterium at C-2. The results indicate the operation of a kinetic isotopic effect k~lk= 2 ~1.33 for malate dehydrogenase, in the given conditions. As expected, this is different from the true intermolecular kH/kzH = 1.85 [4], and depends on the degree of turnover (kH/kzH= 1 if complete). A reduction of transiently formed oxaloacetate by NADH and [4-2H1]NADH, generated during the reaction, will randomize the label at C-2 (not at C-3) of the initially used malate specimens and further decrease the isotopic effect. The apparent kH/kzH= 1.33 was used to exclude racemization at C-3 of the stereospecifically labelled malates at the level of transiently formed oxalacetate as follows. The operation of the isotopic effect must be reflected in the 3H/14Cratios of the initially used malate specimens (3H/14C= 0.787) and the isolated acetate (3H/14C = 1.184). The latter must increase two-fold over the first in the absence of isotopic discrimination and must increase 2/1.33-fold if k ~ / k q , = 1.33. The 3H/’4C ratio of the generated chiral
= NADx [1.2-’H&1 acetaldehyde
[ 1.2- 2H5 lethanol
OH
Generation of Chiral Acetates ,from Stereospec ijiically Labelled Malates
244
Chiral Acetates
Table 3. Formation of stereochemically pure chirul acetate.P In (A) S-acetate was generated in normal water from 2S,3R-[2-ZH1,3-2H1,3-3Hl]maiate [U-14C]malate.In (B) R-acetate was generated in tritiated water, specific activity 9.23 x lo5 counts x min-' x pmol-I, from 2S,3S-[2-2H1,3-2H1]rnalate. In both (A) and (B) unlabelled carrier malate (2.0 pmol) was added after the mixture had been used for acetate production. The isolated acetate Contained about 2.5 pmol ofunlabelled carrier acetate
+
Expt
A
Amount
3H
pmol
counts x mind' (?< initial)
2S,3R-[2-2H1,3-2Hl~H1]Malate [U-14C]Malate Reisolated malate
5.58 0.54 6.42
2.17 x lo6 (100)
-
-
Isolated S-acetate
3.08
1.45 x lo5 (6.7)
2.75 x lo6 (100) 2.23 x lo6 (81) 1.22 x 105 (8.9)
2S,3S-[2-'HI ,3-'H1]Malate Reisolated malate Isolated R-acetate
5.61 5.5 3.06
-
-
4 . 8 lo3 ~ 8 . 8 7 104 ~
-
Substance
~
1.85 x lo6 (85.8) -
B
acetate should therefore be 0.787 x 2/1.33 = 1.184. This was the experimental value found. From the 3H and I4C content of the isolated acetate it can be calculated that at least 0.374 pmol 2S,3R-[2-2H1,3-2H~~Hl]malate and 0.048 pmol [U14C]malate underwent oxidation and subsequent hydrolysis. If corrected for loss of substance from determination of concentration and radioactivity the total is not 0.42 prnol but 0.53 pmol as was determined from pyruvate consumption. The turnover of malate therefore corresponds in fact to 182000 counts 3Hx min-' and 153000 counts 14Cx min-'. Subtraction of these numbers (I4C times two) from the radioactivity of the starting material yields 3H/14C = 0.814 for residual malate. The agreement with the corresponding experimentally determined 3H/14Cratio = 0.835 is 98 %. From these results we conclude that no significant loss of tritium occurred at the level of transiently formed oxaloacetate by configurational instability or at the level of malate by the presence of trace amounts of fumarase. The formation of R-acetate from 2S,3R-[2-'Hl, 3-'Hl]malate in tritiated water was performed likewise, but without ['4C]malate. It was stopped after 0.53 pmol substrate had been consumed as judged from pyruvate consumption. Correcting again for 20 ",:loss of product, the specific activity of the isolated acetate amounts to 2.1 x lo5counts x min-' x pmol-'. The re-isolated malate contained little tritium, about 870 counts x min-' x pmol-I, that is about 0.1 of the specific activity of the tritiated water used. This result strengthens the conclusion that no significant side reaction occurred and hence that stereochemically pure chiral were formed in the outlined conditions. Amplitude of the Assay The configurational assay of the isolated R-acetate and S-acetate was performed as usual to yield tritium
3H/14Cratio
I4C
-
0.787
-
-
Table 4. Loss fumarase Malate derivation
of
tritium f r o m labelled mahtes in the presence of
Time
3H/14Cratios found
average
01 02
03 5 34 45 60
S-Acetate
01 02 03
5 34 45 60 100
cf initial
x
min
R-Acetate
0.835 1.18
1.043 1.099 1.032 0.944 0.854 0.814 0.862 0.953 0.968 1.016 0.287 0.198 0.204 0.194 0.201
1.058
100 f 0.7
0.843
79.7
k 0.7
0.979
100 i 0.9
0.199
20.3 k 0.9
retentions in the fumarase-treated malate specimens of 79.7 and 20.3% respectively (Table 4). The complementarity is important. It would not be expected to arise in using differently labelled m a k e s and solvents if a nonspecific protonation of an oxaloacetate enolate anion had taken place during chiral acetate generation. The data yield an average A 50 % value = 29.74 f 0.6 from which kH/kzH = 3.9 f 0.2 results. The A 50 value = 29.7 was reproduced exactly in a parallel experiment. It is furthermore in agreement with the ~ k 0.5) which was value of 29 f 2.5 % (or k ~ / k =2 3.7 found during another study presented in this paper (stereochemistry of the malate synthase reaction). This value was obtained by a method which yields results independently of the stereochemical purity of the
245
H. Lenz and H . Eggerer Table 5. ConJ&ztional analysis of enzymically prepared clziral acetate The percentage of stereochemical purity was determined by relating the A 50 ”/, value t o the A 50 acetates. Sample
Volume
Time
Isolated acetate
value
Malate turnover
=
29 for stereochemically pure chiral
Configurational Assay A 50 ”/, value
ml
min
counts ’H x min-’
2
0.5 0.3 0.3 0.6
20 45 80 130
120000 163000 170000 373000
42.7 64.0 79.0 92.0
chiral acetates initially used. The average of both determinations of the isotopic discrimination on malate synthase corresponds to k ~ / k = 2 3.8 ~ f 0.1 or, if allowance for counting errors is made, to a A 50% value = 29 & 2%. The conclusions drawn from all these results is that this value represents in fact the actual amplitude of the configurational assay in the given conditions. The chiral acetates synthesized by Cornforth and Redmond by purely chemical means [3,7a] yielded a A 50% value = 26.5 [7a] which is very near to our figure. Considering the steps where ‘racemization’ could occur during chemical synthesis, this high stereochemical purity befits the excellence of this piece of work. Enzymic Preparation qf Clziral Acetates The formation of chiral acetates in the sequence malate + oxaloacetate + acetate as described so far in this paper had the purpose of obtaining stereochemically pure samples. Several precautions such as small malate turnover were taken in these carefully controlled experiments to avoid racemization at the level of transiently formed oxaloacetate. In order to establish a preparative method with high substrate turnover under less controlled conditions it was necessary to show that the chiral acetates generated this way were of satisfactory stereochemical purity. This was checked as follows. 2S,3R-[3-2H1]Malate (4.3 pmol) was incubated in tritiated water (0.35 Ci in a total volume of 2.0 ml 40 mM Tris buffer, pH 8.0, containing 1 mM MnC12) with 10 pmol NAD, 19 U malate dehydrogenase, 13 U oxalacetase and 10 U lactate dehydrogenase. The reaction was followed optically from the increasing NADH concentration and stopped after 130 min incubation time ( 9 2 x malate turnover). NADH formed during the reaction was oxidized from time to time by adding 0.2-pmol samples of pyruvate (4 pmol in total). Samples were withdrawn at the intervals shown in Table 5 and used for the determination of residual malate as well as configurational assay of the acetates generated. The results (Table 5) show that chiral acetates were
purity 0
’
Enzymic Generation of Chiral Acetates. A Quantitative Evaluation of Their Configurational Assay Helmut LENZ and Hermann EGGERER Fachbereich Biologie und Vorklinische Medizin, Universitit Regensburg (Received December 29, 1975 /February 23, 1976)
1. R-Acetate was generated enzymically from R-acetate in the sequence acetate + malate oxaloacetate acetate, and S-acetate likewise from S-acetate. It was concluded that the formation of malate on malate synthase involves the operation of a normal isotopic effect combined with inversion of configuration. The malate synthase k H / k 2 ” was determined as 3.7 -t 0.5 by a method which yields results independently of the stereochemical purity of the chiral acetates used initially. 2. R-Acetate was also generated from R-acetate in the sequence acetate -+ citrate malate oxaloacetate -+ acetate, and S-acetate likewise from S-acetate. The conclusion is the same as given above, but refers to the formation of citrate on the re-synthase. 3. 2S,3R-[2-2H1,3-2H1,3H1]Malate and 2S,3 S-[2-2H1,3-2H~]malate were prepared from 2 S [2,3-2H3]malate by treatment with fumarase in tritiated water and normal water, respectively. It was assumed that these malate specimens were pure with respect to chirality as generated by isotopic labelling. 4. These two malate specimens were partially converted (about 9%) to acetates in conditions where no racemization at the level of transiently formed oxaloacetate occurred. That no racemization took place was demonstrated experimentally. Oxidative enzymic hydrolysis of 2S,3R[2-2H1,3-2H1,3Hl]malate in normal water and of 2S,3S-[2-2H1,3-2H1]malate in tritiated water produced S-[2H1,3Hl]acetate and R- [’HI ,3Hl]acetate, respectively. 5 . The isolated R-[’HI ,’Hl]acetate and S-[2H1,3H1 ]acetate on configurational analysis yielded malates which in the presence of fumarase retained 79.7 k 0.7% and 20.3 f 0.9%, respectively, of their total tritium content. The symmetric deviation from the 50 2, value found with [3Hl]acetate strenghtens the conclusion that stereochemically pure chiral acetates were analyzed. The malate synthase ~ H / ~ Lwas H determined from the data of this study as 3.9 k 0.2. 6. The average of the values given under paragraphs 1 and 5 for the isotopic discrimination on malate synthase corresponds to k ~ / k = 2~ 3.8 & 0.1. I t was concluded that the configurational analysis of stereochemically pure R-[2H1,3Hl]acetate and S-[’HI ,3Hl]acetate yields malates which in the presence of fumarase retain 79 k 27; and 21 k 2”/;;, respectively, of their total tritium content. Hence, a deviation of 29 f 2 ”/c, from the SO:.;’, value represents the actual amplitude of the configurational assay. 7. Outlines are given for an enzymic generation of chiral acetates in preparative scale. -+
-+
-+
Work performed in cooperation with J. W. Cornforth established a configurational assay of asymmetric methyl groups [2,3]. This was achieved by a combination of purely chemical methods for the synthesis of R-[2H1,3Hl]acetate and S-[’H1~H1]-ace~~
These results are taken from the Thesis of H . Lenz, Universitiit Regensburg 1973; a preliminary account of part of this work has appeared elsewhere [I]. EKJ.~WCS. Alcohol dehydrogenase (yeast) (EC 1 . I . I . 1); citrate (r~)syntliase(EC 4.1.3.28); citrate. (si)synthase (EC 4.1.3.7); fuinarase (EC 4.2.1.2); lactate dehydfogenase (EC 1.1.1.27); malate dehydrogenase (EC 1.1.1.37); inalate synthase (EC 4.1.3.2); oxalacetase (EC 3.7.1.1).
-+
tate, and of purely enzymic methods for their configurational determination in the sequence acetate acetyl-CoA + malate -+ fumarate. R-Acetate and glyoxylate on the enzyme malate synthase produced malate which on incubation with fumarase retained more than 5 0 X , whereas malate derived from Sacetate retained less than 50 ?< of its total tritium content. It was thus possible to determine the absolute configuration of chiral acetates generated in any reaction. Although extensive use of the assay has since been made in the elucidation of the substrate stereochemistry of enzyme-catalyzed reactions, that -+
238
Chiral Acetates
of malate synthase remained ambiguous [3]. The outlined experimental results are consistent with inversion of configuration if a normal isotopic effect, kH > kzH, operates on malate synthase. However, these results are also consistent with retention of configuration if this isotopic effect is inverse, kH < k 2 H ; this is rare, but not unknown [4,5]. The pairs of tritium-labelled malate specimens derived from Racetate on operation of either of the mechanisms are shown, respectively, in Eqns (1) and (2).
+
4
[minor)
[ major I
( minor
1
As can be easily visualized, incubation of each of these pairs with fumarase, which catalyzes an antielimination of the elements of water, must yield identical tritium retentions. The stereochemical course of the malate synthase reaction, therefore, remained to be established (see [3] for full discussion). The same factual situation applies for the recitrate synthase reaction where the elucidated substrate stereochemistry, inversion of configuration during methyl-methylene interconversion, also depends on the operation of a normal isotopic effect [6]. Note that the experimental results are consistent only with the outlined ambiguities; other possibilities such as inversion of configuration combined with the operation of an inverse isotopic effect need not be considered. Another unsettled problem concerned the actual amplitude of the configurational assay. This amplitude represents the difference of tritium retention which is found if malates derived on the synthase from stereochemically pure, e.g. R-acetate, and from symmetrically labelled [3H1]acetate are incubated with fumarase. Malate derived from [3Hl]acetate yields the statistically expected 50 %, that derived from R-acetate yields a higher and that from S-acetate a correspondingly lower retention of tritium [3]. This deviation of tritium from the 50% value is designated as the A 50 value thereafter and depends, of course, on the stereochemical purity of the chiral acetates used. It was found initially that R and S acetates produced malates which on incubation with fumarase retained 69 and 31% (A50% value = 19) of their total tritium content, respectively [3]. The corresponding data were 76 and 25% after
the method of chiral acetate preparation was slightly modified [7a]. Independent work of our Swiss colleagues established a A50% value = 40 [8]. Thus, corresponding the amplitude varies from 19- 40 to apparent intramolecular isotopic effects of k ~ / k 2 ~ (or kzH/kH)= 2.2-9.0. Systematic studies have already shown the reliability of the assay; possible sources of error such as optical instability of the chiral acetates or dependence of the malate synthase k ~ / k 2on~ the concentration of acetyl-CoA, have been excluded [7a]. Furthermore, assuming that a A 50 % value = 40 would represent the true amplitude, it can be calculated that our best samples of chemically synthesized chiral acetates would have been ‘racemic’ to the extent of about 35 %. This however, appeared unlikely. A solution for all these problems is possible in principle, if malates stereospecifically labelled at C-3 are converted into chiral acetates. The enzyme oxalacetase which hydrolytically cleaves oxaloacetate to acetate and oxalate was used for this transformation, firstly because oxaloacetate in the presence of NAD and malate dehydrogenase can easily be generated from malate and secondly because the substrate stereochemistry of oxalacetase is now known [9]. The results are shown in this paper and clarify the outlined ambiguities. An enzymic method for the preparation of chiral acetates is presented additionally.
x,
MATERIALS AND METHODS Enzymes and Suhstrates Enzymes, biochemicals, substrates and methods were as described in the preceding communication [9] with exceptions as follows. The 2S,3R-[3-3H1]malate (98 - 99 % monodeuterated [lo]) used for the generation of chiral acetate in preparative scale, the R- [2H1,3H1]acetate and the S-[2H1,3H~]acetate[7a] (specific activity respectively 7.95 x lo6 and 7.7 x lo6 counts x min-‘ x pmol-’) were a gift from Professor J. W. Cornforth ; re-citrate synthase (specific activity 0.8 U/mg protein) [ l l ] was a gift from Professor G. Gottschalk. Hexadeuteroethanol (>98 % *H) and deuterium oxide (99.75%) were from Merck, Darmstadt. Acetyl-CoA was prepared from R-[2H1,3Hl]acetate by the method of Wieland and and S-t2H1,3H~]acetate Bokelmann and used for enzymic synthesis of 2Rcitrates as described [6] (4.0 pinol acetyl-CoA derived from R-acetate yielded 3.64 pmol 2R-citrate of specific activity 3.68 x lo6 counts 3H x min-’ x pmol-’). The isolated citrate was degraded enzymically to the correspondingly labelled malate (3.5 pmol) as described [6]. The results given here and below for R-acetate were obtained likewise with S-acetate.
H. Lenz and H. Eggerer
All cleavage reactions of stereospecifically labelled malate specimens yielding chiral acetates and oxalate were performed in the presence of 3 mM NAD, 1 mM MnC12, malate dehydrogenase, oxalacetase, lactate dehydrogenase and pyruvate. Cleavage of Malate Formed on Malate Synthase
R-['H1:Hl]Acetate (10 pmol; 7.95 x lo6 counts xmin-') was converted to malate [3] and this was isolated (7.63 pmol; 5.09 x lo6 counts x min-I). [U14 CIMalate (0.21 pmol; 6 x lo5 counts x min-') was added to 2.29 pmol(l.53 x lo6 counts x min-') of the R-acetate-derived malate and the mixture was incubated in a total volume of 0.8 ml with 2.2 U oxalacetase (specific activity 6.65 U/mg protein) and 0.18 U malate dehydrogenase. Pyruvate (0.55 pmol ; slight excess) was added within 25 min and the reaction stopped after 30 min. Unlabelled carrier acetate (2.24 pmol) was added and the product acetate isolated by Bartley diffusion with a yield of 0.58 pmol (57900 counts 3 H ~ m i n - 'and 11300 counts I4Cx min-'; 18% conversion of malate; low yield due to a technical error). This was used for configurational analysis. Cleavage of Malate Derived,from 2R-Citvate
The isolated malate (1.4 pmol; 8.3 x lo5 counts 3H x min-') and 0.11 pmol [U-'4C]malate (5.23 x 10' counts x min-') were incubated in a total volume of 1.05 ml with 0.4 U malate dehydrogenase and 4 U oxalacetase (specific activity 7.5 U/mg protein). Pyruvate (0.36 pmol; slight excess) was added within 25 min and the reaction stopped after 40 min. Unlabelled carrier acetate (2.45 pmol) was added and the product acetate was isolated by Bartley diffusion withayieldof1.75pmol(9.18 x lO4counts3Hxmin-' and 3 . 3 4 lo4 ~ counts I4C xmin-'; 17% inalate conversion). This was used for configurational analysis.
PREPARATION OF STEREOCHEMICALLY PURE CHIRAL ACETATES
239
(0.6 mg protein; 650 U) of a commercial crystal suspension were dissolved and lyophilized twice in 1.0 ml 2H20; the residue was dissolved im 0.4 ml 2H20. 0.1 ml alcohol dehydvogenase (yeast) (3 mg protein, 600 U) of a commercial crystal suspension was dissolved and lyophilized twice in 0.4 ml ' H 2 0 and finally dissolved in 0.4 ml 2H20. The reaction mixture contained deuterated compounds in a total volume of 5.5 mI as follows: Tris buffer, 50 pmol; NAD, 10 pmol; hexadeuteroethanol, 1.75 mmol; alcohol dehydrogenase, 0.25 ml; malate dehydrogenase, 0.25 ml and the rest deuterium oxide; the final pH was 7.5. The total oxaloacetate solution was added in four 0.25-ml samples (about 22 pmol) within about 100 min and the reaction was followed at 366 nm with one fifth of the mixture from the increasing NADH concentration. This was observed after the first two oxaloacetate samples had been added but not thereafter. The reaction was stopped by heat treatment (3 min at 100 "C) after 350 min. [U-14C]Malate (18 nmol; 180000 counts x min-') was added; the mixture was desalted by passage through Dowex-50 (H' ; 1 x 5 cm) and 2S-[2,3-2H3]malate was isolated by chromatography on Dowex-1. The isolated malate (48.8 pmol; 1.76 x lo5 counts x min- ') contained about 0.04% [U-'4C]malate, that is an amount which does not influence the production of stereochemically pure chiral acetates.
2S,3S-[2-2H1,3-2H1]Malate
The reaction mixture in a total volume of 1.0 ml contained 19.5 mM 2S-[2-2H1,3-2Hz]malate,70 mM phosphate buffer, pH 7.5, and 50 U fumarase. The reaction was started with the enzyme and followed at 244 nm (fumarate formation) until the equilibrium between malate and fumarate was established (about 1 min). The mixture was then kept at 40°C for 60 min and the reaction stopped by heat treatment (5 min at 100 "C). 2S,3S-[2-2H1,3-2HI]Malate(12.8 pmol; 66% of theoretical value; about 20% are lost as fumarate) was isolated by chromatography on Dowex-I after the solution had been desalted with Dowex-50 (H').
2S-[2,3-2H3]M~l~tr
The deuteration of starting material was performed as follows. 605 mg Tris were dissolved and lyophilized five times in 1.5 ml deuterium oxide and the residue was dissolved finally in 1.65 ml 2H20 to yield a 3 M solution. 19.9mg NAD (about 25 pmol) were dissolved and lyophilized four times in 1.0 ml 2Hz0; the residue was dissolved in 0.5 ml 'H20. 18.5 mg oxaloacetate (1 10 pmol) were dissolved and lyophilized three times in 1.0 ml 2H20; the residue was dissolved in 1.0 ml 2H20 and neutralized to pH 7.3 with 0.11 ml of the deuterated Tris buffer. 60 pl malate dehydrogenase
2 S,3 R-[2-2H1,3-2H~,3H1]Malate
The reaction mixture in a total volume of 1.0 ml contained : 19.5 mM 2S-[2,3-2H3]malate, 70 mM phosphate buffer, pH 7.5, 0.5 ml tritiated water (0.1 Ci) and 50 U fumarase. The reaction was performed and malate was isolated exactly as described above for 2S,3S-[2-2H~,3-2H~ ]malate but without the addition of [14C]malate. The specific activity of the isolated 2S,3R-[2-3H1,3-2H1,3H1]malate (12.4 pmol; 63.5 y: of theory) was 4.32 x lo5 counts x min-'
Chiral Acetates
240 x pmo1-l and that of tritiated water in the incubation mixture was 9.29 x lo5 counts x min-’ x pmol-I. The results indicate within experimental error that malate had been completely equilibrated with tritiated water.
Generution of S-[2Hl,3H1/ Acetate ,from 2 S,3 R - [ ~ - ’ H I , ~HI - ~,3 H ~ ] M u l u t e The reaction mixture in a total of 1.24 ml contained: 5.58 pmol 2S,3R-[2-2Hl,3-2H~ ,3H1]inalate (about 2.4 x lo6 counts 3H x min-’), 0.54 pmol [U‘“Clmalate (about 2.75 x lo6 counts 14C x min-I), 100 pmol Tris buffer, pH 7.0, 3 pmol NAD, 1 pmol MnC12, 10 U lacetate dehydrogenase, 10.5 U oxalacetase (specific activity 10.7 U/mg protein) and 0.5 U malate dehydrogenase. The latter enzyme was used to start the reaction; the initial rate of malate turnover in these conditions was about 15 of that found when malate dehydrogenase was added in excess. The generated NADH (followed at 366 nm) was oxidized during the reaction by adding 0.16 pmol three times and 0.08 pmol pyruvate once. After a total incubation time of 25 min and just before the reaction was stopped (6 min at 100 “C)the absorbance at 366 nm (NADH) started to increase again. Unlabelled carrier material (2.8 pmol each of acetate and pyruvate; 2.0 pmol each of lactate and malate; 5.0 pmol oxalate) were then added and the mixture was desalted by passage through Dowex-50 (H’ ; 1 x 2.5 cm). Acetate and malate were isolated by chromatography on Dowex-1 (X-8, CI-; 1 x 17 cm) with linear HCI gradients [12]:acids were detected by determining radioactivity and conductivity. Malate was eluted (3.2 ml fractions; flux rate 65 ml/h) with a gradient formed between water (200 ml) and 0.01 N HCI (250 ml) and appeared in fractions 84- 103. Acetate was eluted afterwards with a linear gradient formed between 0.07 N HCI (100 ml) and 0.5 HCI (100 ml) in otherwise unchanged conditions (fractions 45 - 58). The malate-containing fractions were combined and evaporated in vacuo; those containing acetate were treated likewise but neutralized with NaOH before evaporation. The re-isolated 2 S ,3 R- [2-2H ,3-2H ]malate (6.42 pmol; 79 yc,of theory with respect to starting material plus carrier) contained 2.23 x lo6 counts I4C x min-’ and 1.85 x 106counts3Hxmin-’. TheisolatedS-[’HI, 3Hl]-acetate (3.08 pmol; about 2.5 pmol unlabelled carrier material) contained 1.22 x lo5 counts ‘“C x min-’ and 1.45 x lo5 counts 3H x min-’. Generution of R-[’H1 ,3H1]Acetate froin 2S,3S-[2-2H~,3-2H1]M~l~te The partial oxidation and subsequent hydrolysis of 2S,3S-[2-ZH1,3-2Hl]malate(5.61 pmol) and the isolation of acetate and residual malate was performed
exactly as described above for the cleavage of the 2S,3 R-[2-2Hl ,3-2H~ ,3H1]malate specimen but in the absence of [I4C]malate and in the presence of tritiated water (specific activity of 3 H H 0 in the incubation mixture: 9.23 x lo5 counts x min-’ x pmol-’). The re-isolated 2S,3S-[2-2Hl,3-2H~ Jmalate (5.5 pmol) contained 870 counts 3H x min-’ x pmol-’. The isolated R-[2H1,3H1]acetate (3.06 pmol; about 2.5 pmol unlabelled carrier acid) contained 8.87 x lo4 counts x min-‘. OTHER METHODS
The configurational analysis of chiral acetates was performed as described elsewhere [3]. Determination of enzymes, substrates and radioactivity was performed as described in the preceding paper [9]. Acetyl-CoA was determined with citrate synthase [13]. The 3H/’4C ratios given in the tables are the average of several determinations of one and the same sample; the percentage deviations were calculated from these determinations as standard error of the means (SE = s/p). This was also used to calculate ‘average A 50%, values’ from the 3H/’4C ratios given in the tables for the malates derived from R-[2Hl,3H1]acetate and S-[’H1 ,3Hl]acetate (usually six independent determinations). Progress of error for z = x/y was calculated as usual from A ; = xAy/y2 Ax/y.
+
RESULTS AND DISCUSSION INVERSION OF CONFIGURATION O N MALATE SYNTHASE A N D re-CITRATE SYNTHASL
The citrate specimens derived from chiral acetylCoA and oxaloacetate on the re-synthase are labelled stereospecifically at the pro-R side chain and can easily be degraded enzymically to the correspondingly C 3labelled malates [6]. Since the stereochemical ambiguities are the same for both the re-synthase and malate synthase reactions, their solution is reducible to one: that is, how to differentiate between the pairs of malate specimens shown in Eqns 1 and 2. If any specimen of malate containing both [3-2H1?Hl]malate and (33 H ]malate ~ is subjected to oxidative hydrolysis in unlabelled water with malate dehydrogenase and oxalacetase, chiral acetate can only be produced from [3-2H1,3Hl]malate. Since the stereochemistry of the hydrolytic reaction is now known [9], assay of the chirality of this acetate defines the chirality at C-3 of the [3-2H1,3H~]malate. If the 3-methylene group of the malate originated by an enzymic reaction from chiral acetate of known chirality, then the stereochemistry of this reaction is also defined. The absolute magnitude of its kinetic deuterium isotope effect can additionally be deduced from the apparent stereo-
H. Lenz and H. Eggerer
24 1
. malate _-dehydrogenase
___
i
(Ql&2;H +
0
I/
(major) retent ion
t (minor)
(major)
f
--
malate dehydrogenase-oxalacetase
(major)
i
(minor]
Scheme 1
chemical purity of the acetate generated by oxidative hydrolysis. The first malate pair (Eqn 1) will produce R-acetate accompanied with a lesser degree of symmetrically labelled [3Hl]acetate, whereas the second (Eqn 2) will produce S-acetate and a larger amount of [3H1]acetate. This is outlined in Scheme 1. Malare Synthase Chiral acetates, Rand S , were converted enzymically to the malates [3]; one part of the isolated malates was analyzed by incubation with fumarase and another part of each sample was cleaved to acetate and oxalate by oxidative hydrolysis. This was performed by incubation with NAD, oxalacetase, pyruvate, lactate dehydrogenase and limiting amounts of malate dehydrogenase as was described previously [9] and the reactions were stopped after about 20 % substrate had been consumed. The acetates were isolated and again analyzed in the sequence acetate -+ malate + fumarate. The procedure is outlined below for Racetate. R-Acetate(l)+malate(R l)+acetate(Ri)
1 fumarate(R 1)
+ malate(R2)
1 fumarate(R 2)
The experimental results are summarized in Table 1 and show that the R and S acetate (1) specimens on direct analysis yielded malates which retained respectively 76.0 and 24.5% of their total tritium content (average A 50"/, value: 25.81 k 0.24). The acetate(2) specimens, derived from R and S acetate in the sequence acetate(1) -+ malate( 1) + acetate(2), yielded
malates(2) which on incubation with fumarase retained 69.5 and 29.00/,, respectively, of their total tritium content (average A 50% value: 20.24 k 0.37). Thus, R-acetate was generated from R-acetate in the overall reaction, and S-acetate likewise from Sacetate. The results demonstrate that a normal isotopic effect operates during formation of malate on the synthase and that the methyl group is converted into the methylene group with inversion of configuration. A comparison of the A 50 "/, values shows that the stereospecific distribution of tritium at C-3 of the R and S acetate-derived malates(2) corresponds to (20.24 f 0.37)/(25.81 k 0.24) x 100 = 78.5 f 2.2% of that of the malate(1) specimens. The degree of 'racemization' follows as 100-78.5 = 21.5%. This means that on synthesis of the malate(1) specimens, 78.5 of the chiral acetates lost hydrogen and 21.5 2 lost deuterium and corresponds to k H / k 2 H = (78.5 2.2)/(21.3 f 2.2) = 3.7 f 0.5. Note that the intramolecular isotopic effect thus determined is independent of the degree of stereochemical purity of the chiral acetates used initially. re-Citrate Synthase On si-citrate synthase acetyl-CoA adds to the siface of oxaloacetate and the methyl-methylene group transformation proceeds with inversion of configuration [7- 7c]. The stereochemical course of the re-synthase is the opposite in that the acetyl group adds to the re-face of oxaloacetate [14]. It was, therefore, of additional interest to see whether evolution produced an enzyme with completely different stereochemistry, that is whether the condensation reaction
242
Chiral Acetates
Table 1. Loss of tritium from luhelled mulutes, derived,frorn chirul acelutes hcJorr and after the intermediate formation of malate, it1 the presence of fumarase
Table 2. Loss of tritium from labelled malutrs, derived from 2Rcitrutes und f i o m 2 R-ritrutr-derived mulutes, in the presence of famaruse
Malate derivation
Malate derivation via citrate
Time
'H/14C ratios found
average
01 02 03 35 45 60
S-Acetate( 1)
01
02
0.3 35 45 60
Acetate(R2)
01 02 03
5 25 40 60
Acetate(S 2)
01 02
03 5 25 40 60
initial
x
min R-Acetate
c/.
1.801 1.794 1.786 1.376 1.347 1.36X 1.751 1.765 1.753 0.429 0.427 0.435 0.905 0.902 0.901 0.687 0.623 0.627 0.632 0.756 0.760 0.765 0.384 0.222 0.218 0.221
found
100 f 0.2
01 02 03
1.364
27 45 62
76.0 rfr 0.7
Acetate(S 1) 1.765
0.430
0.903
100
k 0.3
*
01 02
03 27 45 62
24.5 rfr 0.6
100
Acetate(R2) 0.2
01 02
03 5
0.627
69.5
i 0.4
0.760
100
0.4
25 40 60
Acetate( S 2)
01
02 03
0.220
'H/14C ratios average
29.0 rfr 0.6
5 25 40 60
cf. initial
x
inin Acetate(f7 1)
1.794
Time
0.342 0.347 0.336 0.225 0.224 0.227 0.434 0.439 0.432 0.146 0.148 0.152 0.656 0.664 0.655 0.537 0.441 0.430 0 439 0.520 0.506 0.520 0.288 0.175 0.164 0.176
0.342
100
k 0.6
0.225
65.8
k 0.4
0.435
100
k 0.4
0.149
34.3 f 0.9
0.658
100 rfr 0.3
0.437
66.4 rfr 0.6
0.515
300 f 0.4
0.172
33.4
k 0.9 ~
above, on incubation with fumarase, yielded tritium retentions of 66.4 and 33.3 respectively (average A 50% value = 16.51 0.40). The results establish that R-acetate was generated from R-acetate in the overall sequence and S-acetate likewise from S-acetate. Thus, the formation of citrate on the re-synthase as well as on the si-synthase involves the operation of a normal isotopic effect and proceeds with inversion of configuration during methyl-methylene group transformation. The chiral acetates used in this study on direct configurational assay produced an average A 50% value of 25.81 f 0.24 (data from Table 1); they yielded citrate derived malates(2) via malates(c) of average Acetate(1) -+ citrate --t malate(c)+acetate(2) + malate(2) A 50"/, value = 16.51 f 0.40. On synthesis of citrate (16.51 0.40)/(25.81 f 0 . 2 4 ) ~100 = 64.0 f 2.1'x 1 1 1 fumarate( 2) of the acetate( 1) specimens therefore lost hydrogen malate(1) + fumarate(1) fumarate(c) and 36.0 & 2.1 lost deuterium (kH/kzH = 1.8 & 0.2). This determination of the intramolecular isotopic The experimental results are summarized in TableZ effect for the re-synthase is again independent of The malates(c) derived from the acetates(1) via the stereochemical purity of the chiral acetates used citrate yielded an average A 50 %,value of 15.86 k0.2 '%;. This provides no new information but is in agreement initially. Other determinations, e.g. from the data for malates(c) in Table 2, which depend on this with previous results [6]. The malate(2) specimens obtained from R and S acetate in the sequence given stereochemical purity are in satisfactory agreement. on the re-synthase proceeded with retention of configuration. Chiral acetates were converted into citrates on the re-synthetase and the isolated citrates were degraded to the malates(c) by incubation with citrate lyase, NADH and malate dehydrogenase [6]. The expected malate specimens are qualitatively those shown in Scheme 1 for their formation from chiral acetyl-CoA and glyoxylate on malate synthase. The malates(c) were subjected to the oxalacetase reaction and the thus-generated acetates(2) were analyz by the configurational assay as usual. The overall procedure consists of the steps shown below.
x)
H. Lenz and H. Eggerer
243
If corrected for the presence of about 10 % ‘racemic’ material in the initial acetates, the data for malates(c) in Table 2 yield kH/kzH= 2.1. It remains to be noted that the A 50% value for the malates(c) would be zero and that for the malate(2) specimens about 14.5 if no isotopic discrimination occurred during formation of citrate on the re-synthase. AMPLITUDE OF THE CONFIGURATIONAL ASSAY
Preparation Stereospecfically Labelled Malates
of
2S,3R- [2-3HL,3-2H1,3Hl]malate and 2S,3S-[22Hl,3-2Hl]nialate were prepared enzymically from oxaloacetate as follows ( c f [lo]). Hexadeuteroethanol was incubated in deuterium oxide with alcohol dehydrogenase and NAD for formation of 4R-[4-’Hl]NADH. Malate dehydrogenase and [3-2H2]oxaloacetate were also present and the generated 4R[4-IH1]NADH was used in situ for formation of 2S-[2,3-’H3]malate with regeneration of NAD. Considerable experimental precaution was taken to ensure a deuterium content of greater than 99% in each of the three labelled positions of the product malate. The isolated trideuterated malate was incubated with fumarase in either tritiated or normal water for formation of 2S,3 R-[2-2H1,3-2H1,3Hl]malate and 2S, 3S-[2-2H1,3-2H1]malaterespectively, and the products were isolated. The procedure is shown in Scheme 2. (alcohol dehydragenasel
LR-IL-~HII NADH
lrnalate dehyjragenase)
\ H02C’
‘H
/C02H
.*‘
2H/ c-c,
’$H
These malate specimens were partially converted (about 9%) to acetates of opposite chirality by incubation in either normal or tritiated water in the presence of NAD with limiting amounts of malate dehydrogenase and excess oxaloacetase. The concentration of oxaloacetate which decreases rapidly in this coupled assay was restored by adding lactate dehydrogenase and titrating NADH with pyruvate as shown below. Malate
Oxaloacetate
NAD
NADH
Lactate
Pyruvate
H20
i i
R
\\
C
(fumarasel
2H
H02C’
C02H
C ‘ =/ H02C/
\2H
I
1_ _ _ _ _ -
[ oxalacetasel------
I C02H
‘C2H2C02H
Acetate + Oxalate
This operation ensured fast turnover of oxaloacetate and thus helped to avoid its racemization. The generated chiral acetates and the residual malates were isolated and their radioactivity determined. The results are sumarized in Table 3 and allow conclusions as follows, In the formation of S-acetate, from the distribution of radioactivity in the isolated acetate, the conversion of [14C]malate can be calculated as at least 8.9%, and that of [3H]malate as at least 6.7%. Thus, [U-14C]malate containing hydrogen at C-2 reacts about 1.33 times faster than 2S,3R-[2-2H1,3-2H1?H~]malate containing deuterium at C-2. The results indicate the operation of a kinetic isotopic effect k~lk= 2 ~1.33 for malate dehydrogenase, in the given conditions. As expected, this is different from the true intermolecular kH/kzH = 1.85 [4], and depends on the degree of turnover (kH/kzH= 1 if complete). A reduction of transiently formed oxaloacetate by NADH and [4-2H1]NADH, generated during the reaction, will randomize the label at C-2 (not at C-3) of the initially used malate specimens and further decrease the isotopic effect. The apparent kH/kzH= 1.33 was used to exclude racemization at C-3 of the stereospecifically labelled malates at the level of transiently formed oxalacetate as follows. The operation of the isotopic effect must be reflected in the 3H/14Cratios of the initially used malate specimens (3H/14C= 0.787) and the isolated acetate (3H/14C = 1.184). The latter must increase two-fold over the first in the absence of isotopic discrimination and must increase 2/1.33-fold if k ~ / k q , = 1.33. The 3H/’4C ratio of the generated chiral
= NADx [1.2-’H&1 acetaldehyde
[ 1.2- 2H5 lethanol
OH
Generation of Chiral Acetates ,from Stereospec ijiically Labelled Malates
244
Chiral Acetates
Table 3. Formation of stereochemically pure chirul acetate.P In (A) S-acetate was generated in normal water from 2S,3R-[2-ZH1,3-2H1,3-3Hl]maiate [U-14C]malate.In (B) R-acetate was generated in tritiated water, specific activity 9.23 x lo5 counts x min-' x pmol-I, from 2S,3S-[2-2H1,3-2H1]rnalate. In both (A) and (B) unlabelled carrier malate (2.0 pmol) was added after the mixture had been used for acetate production. The isolated acetate Contained about 2.5 pmol ofunlabelled carrier acetate
+
Expt
A
Amount
3H
pmol
counts x mind' (?< initial)
2S,3R-[2-2H1,3-2Hl~H1]Malate [U-14C]Malate Reisolated malate
5.58 0.54 6.42
2.17 x lo6 (100)
-
-
Isolated S-acetate
3.08
1.45 x lo5 (6.7)
2.75 x lo6 (100) 2.23 x lo6 (81) 1.22 x 105 (8.9)
2S,3S-[2-'HI ,3-'H1]Malate Reisolated malate Isolated R-acetate
5.61 5.5 3.06
-
-
4 . 8 lo3 ~ 8 . 8 7 104 ~
-
Substance
~
1.85 x lo6 (85.8) -
B
acetate should therefore be 0.787 x 2/1.33 = 1.184. This was the experimental value found. From the 3H and I4C content of the isolated acetate it can be calculated that at least 0.374 pmol 2S,3R-[2-2H1,3-2H~~Hl]malate and 0.048 pmol [U14C]malate underwent oxidation and subsequent hydrolysis. If corrected for loss of substance from determination of concentration and radioactivity the total is not 0.42 prnol but 0.53 pmol as was determined from pyruvate consumption. The turnover of malate therefore corresponds in fact to 182000 counts 3Hx min-' and 153000 counts 14Cx min-'. Subtraction of these numbers (I4C times two) from the radioactivity of the starting material yields 3H/14C = 0.814 for residual malate. The agreement with the corresponding experimentally determined 3H/14Cratio = 0.835 is 98 %. From these results we conclude that no significant loss of tritium occurred at the level of transiently formed oxaloacetate by configurational instability or at the level of malate by the presence of trace amounts of fumarase. The formation of R-acetate from 2S,3R-[2-'Hl, 3-'Hl]malate in tritiated water was performed likewise, but without ['4C]malate. It was stopped after 0.53 pmol substrate had been consumed as judged from pyruvate consumption. Correcting again for 20 ",:loss of product, the specific activity of the isolated acetate amounts to 2.1 x lo5counts x min-' x pmol-'. The re-isolated malate contained little tritium, about 870 counts x min-' x pmol-I, that is about 0.1 of the specific activity of the tritiated water used. This result strengthens the conclusion that no significant side reaction occurred and hence that stereochemically pure chiral were formed in the outlined conditions. Amplitude of the Assay The configurational assay of the isolated R-acetate and S-acetate was performed as usual to yield tritium
3H/14Cratio
I4C
-
0.787
-
-
Table 4. Loss fumarase Malate derivation
of
tritium f r o m labelled mahtes in the presence of
Time
3H/14Cratios found
average
01 02
03 5 34 45 60
S-Acetate
01 02 03
5 34 45 60 100
cf initial
x
min
R-Acetate
0.835 1.18
1.043 1.099 1.032 0.944 0.854 0.814 0.862 0.953 0.968 1.016 0.287 0.198 0.204 0.194 0.201
1.058
100 f 0.7
0.843
79.7
k 0.7
0.979
100 i 0.9
0.199
20.3 k 0.9
retentions in the fumarase-treated malate specimens of 79.7 and 20.3% respectively (Table 4). The complementarity is important. It would not be expected to arise in using differently labelled m a k e s and solvents if a nonspecific protonation of an oxaloacetate enolate anion had taken place during chiral acetate generation. The data yield an average A 50 % value = 29.74 f 0.6 from which kH/kzH = 3.9 f 0.2 results. The A 50 value = 29.7 was reproduced exactly in a parallel experiment. It is furthermore in agreement with the ~ k 0.5) which was value of 29 f 2.5 % (or k ~ / k =2 3.7 found during another study presented in this paper (stereochemistry of the malate synthase reaction). This value was obtained by a method which yields results independently of the stereochemical purity of the
245
H. Lenz and H . Eggerer Table 5. ConJ&ztional analysis of enzymically prepared clziral acetate The percentage of stereochemical purity was determined by relating the A 50 ”/, value t o the A 50 acetates. Sample
Volume
Time
Isolated acetate
value
Malate turnover
=
29 for stereochemically pure chiral
Configurational Assay A 50 ”/, value
ml
min
counts ’H x min-’
2
0.5 0.3 0.3 0.6
20 45 80 130
120000 163000 170000 373000
42.7 64.0 79.0 92.0
chiral acetates initially used. The average of both determinations of the isotopic discrimination on malate synthase corresponds to k ~ / k = 2 3.8 ~ f 0.1 or, if allowance for counting errors is made, to a A 50% value = 29 & 2%. The conclusions drawn from all these results is that this value represents in fact the actual amplitude of the configurational assay in the given conditions. The chiral acetates synthesized by Cornforth and Redmond by purely chemical means [3,7a] yielded a A 50% value = 26.5 [7a] which is very near to our figure. Considering the steps where ‘racemization’ could occur during chemical synthesis, this high stereochemical purity befits the excellence of this piece of work. Enzymic Preparation qf Clziral Acetates The formation of chiral acetates in the sequence malate + oxaloacetate + acetate as described so far in this paper had the purpose of obtaining stereochemically pure samples. Several precautions such as small malate turnover were taken in these carefully controlled experiments to avoid racemization at the level of transiently formed oxaloacetate. In order to establish a preparative method with high substrate turnover under less controlled conditions it was necessary to show that the chiral acetates generated this way were of satisfactory stereochemical purity. This was checked as follows. 2S,3R-[3-2H1]Malate (4.3 pmol) was incubated in tritiated water (0.35 Ci in a total volume of 2.0 ml 40 mM Tris buffer, pH 8.0, containing 1 mM MnC12) with 10 pmol NAD, 19 U malate dehydrogenase, 13 U oxalacetase and 10 U lactate dehydrogenase. The reaction was followed optically from the increasing NADH concentration and stopped after 130 min incubation time ( 9 2 x malate turnover). NADH formed during the reaction was oxidized from time to time by adding 0.2-pmol samples of pyruvate (4 pmol in total). Samples were withdrawn at the intervals shown in Table 5 and used for the determination of residual malate as well as configurational assay of the acetates generated. The results (Table 5) show that chiral acetates were
purity 0
’
