Malate synthase

FIGURE 20.28 The glyoxylate cycle. The first two steps are identical to TCA cycle reactions. The third step bypasses the C09-evolving steps of the TCA cycle to produce snc-cinate and glyoxylate. The malate synthase reaction forms malate from glyoxylate and another acetyl-CoA. The result is that one torn of the cycle consumes one oxaloacetate and two acetyl-CoA molecnles bnt produces two molecnles of oxaloacetate. The net for this cycle is one oxaloacetate from two acetyl-CoA molecnles. 

The enzyme malate synthase also synthesises (S )-malic acid from glyoxylic acid (OHC-CO2H) and the thioester acetyl-coenzyme A (CH3CO.SC0A this may be regarded as a source of the nucleophile CH2CO.SC0A). 

This procedure involves the enzymatic conversion of the chiral acetate (C(HDT)COO ), obtained from experiments involving chiral methyl groups, to labeled malate. The enzymes used in this procedure include acetate kinase, phosphotransacetylase, malate synthase, and fumarase. 

ABORTIVE COMPLEXES MALATE DEHYDROGENASE MALATE SYNTHASE (S)-2-METHYLMALATE DEHYDRATASE Malate condensing enzyme,... 

MALATE SYNTHASE MALATE DEHYDROGENASE ABORTIVE COMPLEXES Malate dehydrogenase (acceptor),... 

The glyoxylate cycle is active in the germinating seeds of some plants and in certain microorganisms that can live on acetate as the sole carbon source. In plants, the pathway takes place in glyoxysomes in seedlings. It involves several citric acid cycle enzymes and two additional enzymes isocitrate lyase and malate synthase. 

The second substrate glyoxylate approaches from the other side of the molecule and condenses as is shown. Since any one of the three protons in either R or S chiral acetyl-CoA might have been abstracted by base B, several possible combinations of isotopes are possible in the L-malate formed. One of the results of the experiment using chiral (R) acetyl-CoA is illustrated in Eq. 13-43. The reader can easily tabulate the results of removal of the 2H or 3H. However, notice that if the base -B removes 2H (D) or 3H (T) the reaction will be much slower because of the kinetic isotope effects which are expected to be Hk/"k 7 and "k/ k = 16. A second important fact is that the pro-R hydrogen at C-3 in malate is specifically exchanged out into water by the action of fumarate hydratase. From the distribution of tritium in the malate and fumarate formed using the two chiral acetates, the inversion by malate synthase was established. See Kyte231 for a detailed discussion. 

A key enzyme in the glyoxylate cycle is isocitrate lyase, which cleaves isocitrate (Eq. 13-40) to succinate and glyoxylate. The latter is condensed with a second acetyl group by the action of malate synthase (Eq. 13-38). The L-malate formed in this reaction is dehydrogenated to the regenerating substrate oxalo-... 

R and S isomers of HDT]acetic acid were synthesized by chemical and enzymatic methods that yield products of known stereochemistry.1819 The two isomers were then distinguished by using the following ingenious enzymatic assays. The acetic acid was first converted to acetyl-coenzyme A (by a reaction of the carboxyl group—and not the methyl—of acetic acid). The acetyl-coenzyme A was then condensed with glyoxylate to form malate in an essentially irreversible reaction catalyzed by malate synthase (equation 8.27). The crucial feature of this reaction is that it is subject to a normal kinetic isotope effect, so that more H than D... 

It has since been proved that malate synthase proceeds with inversion independently of any assumption about isotope effect. This has been done via a lyase system that splits malate to acetate21—a methylene-to-methyl conversion. By using methylene-labeled malate and generating chiral acetate, it could be shown unambiguously that this cleavage is an inversion. When the lyase reaction was applied to malate formed from chiral acetate by malate synthase, it was found that the acetate thus generated was of the same chirality as the starting material. This proved that there is also inversion in the reaction of malate synthase. 

It should be noted that although the stereochemistry of the products of the malate synthase reaction is now known (see the next section), with the kinetic isotope effect taken into account, this information is not necessary for the chirality assay all that is essential is that R and S isomers of [HDTJacetic acids give different yields of radioactive fumarate.20... 

Topic LI). Thus, animals cannot convert fatty acids into glucose. In contrast, plants have two additional enzymes, isocitrate lyase and malate synthase, that enable them to convert the carbon atoms of acetyl CoA into oxaloacetate. This is accomplished via the glyoxylate pathway, a route involving enzymes of both the mitochondrion and the glyoxysome, a specialized membranous plant organelle. 

Isocitrate lyase and malate synthase. Isocitrate lyase catalyzes the cleavage of isocitrate to succinate and glyoxylate malate synthase catalyzes the condensation of glyoxylate and acetyl-CoA to yield malate. 

While the syntheses described here should produce chiral acetate samples, methods are needed to prove the existence of chirality and to determine its extent. The first method was devised independently by the Comforth and Arigoni groups and is still widely used [122,128,129]. It depends on the use of two enzymes, malate synthase and fumarase. Thus, overall, the sample of acetic acid is first converted to its CoA derivative (either with acetate kinase and phosphotransacetylase or chemically with ClCOOC2H5 and Co ASH) and then to malate and fumarate ... 

The method relies on an isotope effect so that H is removed in preference to 2H and 3H in the malate synthase reaction. In other words, the hydrogen isotope which is eliminated (in formation of the methylene group of malate) is determined by an intramolecular isotope effect such effects normally follow the sequence kH >k2H > k3H. Whether 3H is located in the pro-R or pro-S position of malate is then determined from the known selectivity of fumarase. 

The species, 152, derived by elimination of 3 H is of no practical interest. Tritium is only present at tracer levels so there are large numbers of CH22HCOOH molecules also present these will give rise to 152 by elimination of H. As for 150 and 151, if the malate synthase shows a normal isotope effect ( H > k2H), 150 will predominate over 151. 

After equilibration with fumarase, the mixture of fumarate and malate is analyzed for 3H. If tritium is originally present in the pro-3S position, the equilibration will not remove it (150 plus 152). On the other hand, tritium in the pro-3R position will be lost to the water by way of 153. To facilitate the analysis, [14C]acetate is added initially and the 3H I4C ratio is determined on the malate produced by malate synthase, before and after incubation with fumarase. The tritium content of the incubation water is also determined. The % retention of 3H in the fumarase reaction is given the symbol, F [127]. In detailed analyses, it has been shown that chirally pure (7 (-acetate is characterized by F = 79 and (S (-acetate by F= 21 [129]. The F value actually depends on four factors ... 

---