Claisen enzymes mechanisms

The conversion of [49] into [50] involves a Claisen rearrangement. Once this was realized it was less surprising that no specific catalytic groups on the enzyme are involved. Support for the Claisen-type mechanism comes from the inhibition shown by the bicyclic dicarboxylate [51], prepared by Bartlett and Johnson (1985) as an analogue of the presumed transition state [52], This same structure [51], coupled through the hydroxyl group to a small protein, was used as a hapten to induce antibodies, one (out of eight) of which mimics the behaviour of chorismate mutase, albeit less efficiently (Table 7). 

Walsh refers to the enzymes described in this section (which catalyze carbonyl condensation reactions involving one component that is not an aldehyde or ketone) as Claisen enzymes (6). I shall use that terminology here, although these reactions are not formally Claisen condensations, but they are distinguished by their mechanisms from the other adolases mentioned in Section III,A, 1. 

Enol Mechanism for Claisen Enzymes. An alternative mechanism for Claisen enzymes involves initial conversion of the nucleophilic substrate into the corresponding enol. In this case, addition of the carbon of the enol to the carbonyl of the electrophilic reactant can be assisted by acid-base catalysis. Application of the mechanism to the malate synthase reaction is shown in the Scheme... 

Enzymes catalyze the formation of carbon-carbon bonds between allylic and homoallylic pyrophosphate species by mechanisms that are very different from those for carbonyl compounds. Here, carbonium ions, stabilized as ion pairs and generated from allylic pyrophosphates, are likely to be the intermediates that add to the TT-electron density of carbon-carbon double bonds to form new carbon-carbon single bonds. Reaction patterns are consistent with model systems and the mechanisms are based on analogies with the models, stereochemical information (which is subject to interpretation), and the structural requirements for inhibitors. Detailed kinetic studies, including isotope effects, which provide probes in the aldolase and Claisen enzymes discussed in Section II, have not yet been performed in these systems. The possibility for surprising discoveries remains and further work is needed to confirm the proposed mechanisms and to generalize them. 

Kuo and Rose showed that the proton that is removed is retained by the enzyme (67). Stubbe and Abeles prepared an alternative substrate in which fluoride elimination competes with carboxylation 68, 69). Neither result defines the mechanism, but they do show that it is likely that the carbanion derived from the substrate is generated as an intermediate and therefore the reaction is not concerted. Definitive results come from double-isotope fraction studies by O Keefe and Knowles (70) and by Cleland and co-woricers (71). As described for Claisen enzymes, this methodology tests whether processes occur in one or two steps. Labeling of the carboxyl to be transferred with carbon-13 and the proton to be transferred as deuterium provided the means to do this test. The results indicate clearly that proton removal from the substrate to generate the carbanion and transfer of the carboxyl occurs in distinct steps. The resulting attack of the carb-... 

In this contribution, we describe work from our group in the development and application of alternatives that allow the explicit inclusion of environment effects while treating the most relevant part of the system with full quantum mechanics. The first methodology, dubbed MD/QM, was used for the study of the electronic spectrum of prephenate dianion in solution [18] and later coupled to the Effective Fragment Potential (EFP) [19] to the study of the Claisen rearrangement reaction from chorismate to prephenate catalyzed by the chorismate mutase (CM) enzyme [20]. 

Nature gives us some illustrative examples of iterative methodologies in its biochemical mechanisms. The fatty acid-polyketide biosynthesis is one of them. The assembly of acyl units by sequential Claisen-type condensations to form a polyketide or fatty acid takes place at a multi-enzyme complex, at which the initial molecule is lengthened by one C2-unit per pass of a reaction cycle (Fig. 2). 

Chorismate mutase catalyzes the Claisen rearrangement of chorismate to prephenate at a rate 106 times greater than that in solution (Fig. 5.5). This enzyme reaction has attracted the attention of computational (bio)chemists, because it is a rare example of an enzyme-catalyzed pericyclic reaction. Several research groups have studied the mechanism of this enzyme by use of QM/MM methods [76-78], It has also been studied with the effective fragment potential (EFP) method [79, 80]. In this method the chemically active part of an enzyme is treated by use of the ab initio QM method and the rest of the system (protein environment) by effective fragment potentials. These potentials account... 

Other Claisen condensations are involved in synthesis of fatty acids and polyketides217 (Chapter 21) and in formation of 3-hydroxy-3-methylglutaryl-CoA, the precursor to the polyprenyl family of compounds (Chapter 22). In these cases the acetyl group of acetyl-CoA is transferred by a simple displacement mechanism onto an -SH group at the active site of the synthase to form an acetyl-enzyme.218 219 The acetyl-enzyme is the actual reactant in step b of Eq. 17-5 where this reaction, as well as that of HMG-CoA lyase, is illustrated. 

Figure 1 In a QM/MM calculation, a small region is treated by a quantum mechanical (QM) electronic structure method, and the surroundings treated by simpler, empirical, molecular mechanics. In treating an enzyme-catalysed reaction, the QM region includes the reactive groups, with the bulk of the protein and solvent environment included by molecular mechanics. Here, the approximate transition state for the Claisen rearrangement of chorismate to prephenate (catalysed by the enzyme chorismate mutase) is shown. This was calculated at the RHF(6-31G(d)-CHARMM QM-MM level. The QM region here (the substrate only) is shown by thick tubes, with some important active site residues (treated by MM) also shown. The whole model was based on a 25 A sphere around the active site, and contained 4211 protein atoms, 24 atoms of the substrate and 947 water molecules (including 144 water molecules observed by X-ray crystallography), a total of 7076 atoms. The results showed specific transition state stabilization by the enzyme. Comparison with the same reaction in solution showed that transition state stabilization is important in catalysis by chorismate mutase78.

BPS catalyzed the stepwise condensation of benzoyl-CoA with three molecules of malonyl-CoA to give a tetraketide intermediate that was cyclized by intramolecular Claisen condensation into 2,4,6-trihydroxybenzophenone (Figure 2). The enzyme was inactive with CoA-linked ciimamic acids such as 4-coumaroyl-CoA, the preferred starter substrate for chalcone synthase (CHS). BPS and CHS from H. androsaemum cell cultures shared 60.1% amino acid sequence identity. CHS is ubiquitous in higher plants and the prototype enzyme of the type III PKS superfamily (1,2). It uses the same reaction mechanism like BPS to form 2, 4,4, 6 -tetrahydroxychalcone, the precursor of flavonoids (Figure 2). 

The enzyme could be inactivated by NaBH4 in the presence of either acetoacetyl-CoA or acetyl-CoA. This observation strongly suggests that the reaction is through a Claisen condensation with an amine as the base and with an enzyme substrate ketimine as an intermediate [20]. A mechanism was postulated for the reaction as indicated in Fig. 2. 

Because Claisen condensing enzymes form an integral part of the fatty acid or polyketide biosynthesis, the reader is referred to the relevant chapters of this series for an in-depth discussion of the KAS and PKS enzymes. Several excellent reviews of Claisen-type enzymes have also been published recently and provide detailed perspectives on the mechanism, inhibition, and structural aspects of the thiolase superfamily (see Chapters 1.05 and 1.02). 

Scheme 10 General reaction mechanism of the CoA-dependent Claisen-type condensing enzymes, malate synthase, a-isopropylmalate synthase, citrate synthase, and homocitrate synthase.

There are, however, clear stereomechanistic differences between these two classes of enzyme-catalyzed reactions. The Claisen-type condensations uniformly involve inversion of configuration at the a-carbon of the esteratic substrate, involving C-C bond formation at either the re or the si face of the ketonic or aldehydic substrate (Table VII) (196-211). Moreover, neither Schiff bases nor metal ions have been directly implicated in the catalytic mechanisms of these enzymes. Unlike the aldolases, these enzymes do not catalyze rapid enolization of the nucleophilic substrate in the absence of the second substrate. Inversion of configuration suggests that at least two catalytic groups, perhaps operating in concert, facilitate C-C bond formation. Physicochemical measurements on citrate synthase are consistent with this interpretation of inversion of configuration. 

In all the acetyl coenzyme-A-utilising enzymes which catalyse Claisen-type condensations the reaction involves the conversion of the acetyl methyl into a methylene group. A simple example illustrates the use of thiol esters both as carbanion-stabilis-ing systems and as readily hydrolysable esters. The conversion of glyoxalate to malate uses acetyl coenzyme. A probable mechanism is outlined below ... 

Chorismate mutase (CM) catalyzes the Claisen rearrangement of chorismate to prephenate in the shikimic acid pathway used in the biosynthesis of aromatic amino acids. It represents a reference enzyme to explore the fundamentals of catalysis and has been the subject of extensive experimental and computational research. These have shown both that catalysis proceeds without covalent binding of the substrate to the enzyme, and that the uncatalyzed reaction in water proceeds by the same mechanism. This makes CM a particularly convenient target for QM/MM studies. 

Such a condensation is mediated by the enzyme 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS). The mechanism of this catalysis is outlined in Figure 1.21. An initial frani-thioesterase step transfers the acetyl group of the first acetyl-CoA to an enzymatic cysteine. In the Claisen condensation phase of the reaction, the a-carbon of a second acetyl-CoA is deprotonated, forming an enolate. The enolate carbon attacks the electrophilic thioester carbon, forming a tetrahedral intermediate which quickly collapses to expel the cysteine thiol [22]. 

In the Claisen condensation catalyzed by the enzyme thiolase, acetyl-CoA is converted to its enolate anion, which then attacks the carbonyl group of a second molecule of acetyl-CoA to form a tetrahedral carbonyl addition intermediate. Collapse of this intermediate by the loss of CoAnSH gives acetoacetyl-CoA. The mechanism for this condensation reaction is exactly the same as that of the Claisen condensation (Section 15.3A) ... 

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