Isomerases enolate intermediates

The 500-residue subunits of pyruvate kinase consist of four domains,891 the largest of which contains an 8-stranded barrel similar to that present in triose phosphate isomerase (Fig. 2-28). Although these two enzymes catalyze different types of reactions, a common feature is an enolic intermediate. One could imagine that pyruvate kinase protonates its substrate phosphoenolpyruvate (PEP) synchronously with the phospho group transfer (Eq. 12-42). However, the enzyme catalyzes the rapid conversion of the enolic form of pyruvate to the oxo form (Eq. 12-43) adding the proton sterospecifically to the si face. This and other evidence favors the enol as a true intermediate... 

An interesting use is made of addition to a double bond by glutathione-dependent cis-tmns isomerases.76 One of them converts maleate to fumarate with a turnover number of 300 s"1. Similar enzymes, which participate in bacterial breakdown of aromatic compounds (Fig. 25-7), isomerize maleylacetoacetate and maleylpyruvate to the corresponding fumaryl derivatives (Eq. 13-20). The - SH group of bound glutathione is thought to add to the double bond. Rotation can then occur in the enolic intermediate. Thiocyanate ion catalyzes the isomerization of maleic acid nonenzy-matically, presumably by a similar mechanism. 

In addition to serving as structural motifs, enols and enolates are involved in diverse biological processes. Several enol/enolate intermediates have been proposed to be involved in glycolysis (Section IV.A), wherein c/ -enediol 21 is proposed to be an intermediate in the catalytic mechanism of phosphohexose isomerase and an enol-containing enamine intermediate (22) has been proposed in the catalytic pathway of class I aldolase. In the case of glucose-fructose (aldose-ketose) isomerization, removal of the proton on Cl-OH produces the aldose while deprotonation of C2-OH yields the ketose, which is accompanied by protonation at the C2 and Cl positions, respectively. There are several cofactors that are involved in various biological reactions, such as NAD(H)/NADP(H) in redox reaction and coenzyme A in group transfer reactions. Pyridoxal phosphate (PLP, 23) is a widely distributed enzyme cofactor involved in the formation of a-keto acids, L/D-amino... 

J -3-Keto isomerase catalyzes the isomerization of J -3-ketosteroids to zl -3-ketosteroids by stereospecific transfer of a hydrogen atom from C(4) to C(6). There is considerable evidence that it is the 40- and 6/5-hydrogens that are involved and that the reaction proceeds via an enolic intermediate. A low resolution (6 A) crystal structure determination has been published and the probable steroid-binding site identified via a bound inhibitor, 4-acetoxy-mercuric estradiol. The results of a higher resolution study (2.5 A) combined with the results of NMR studies and analysis of activity of mutant forms of the enzyme have helped to further define the probable active site of the enzyme [64]. 

The different conformations of the 4-ene-3-one A-rings of many hormonal steroids have been discussed in Section 3.2 (see Fig. 3.2.3). A 5-ene-3-one isomerase produces these steroids by a stereospecific transfer of a hydrogen atom from C4 to C6. The 4 - and 6P hydrogens are involved, and the reaction proceeds via an enolic intermediate. The isomerase is an elongated dimer in which the steroid is entrapped in a barrel of eight 3-strands (Fig. 9.6.4). 

Considerable information is available on the molecular properties of this enzyme, on the catalytic mechanism, and on the stereochemistry of the enzymic reaction. The isomerase is composed of identical associating subunits. The primary structure is known and comprises 125 residues (MW 13,394) including all the common amino acids except cysteine and tryptophan. The enzyme, which exhibits exceptionally high catalytic activity, has a molecular activity at saturating concentrations of A -androstene-3,17-dione of 4.38 X 10 min per monomer at pH 7.0 and 25°. Mechanistic studies have disclosed that the isomerase catalyzes the reaction of A < -3-ketostcroids by a direct, stercospccific, and intramolecular transfer of the 4 8-proton to the 6)8 position. There is considerable evidence for the involvement of an enolic intermediate in the... 

Based on the proposed molecular mechanism of this reaction, a series of acetylenic 5,10-secosteroids has been prepared in the belief that they might serve as substrates for A -3-ketosteroid isomerase. Abstraction of the proton at C-4 by the enzyme should then generate, via an enolic intermediate, the corresponding highly reactive conjugated allenic ketones, which might be expected to react covalently with a nucleophilic amino acid residue at the active site (Scheme 3). This proposal was based on expected conformational similarities between the acetylenic 5,10-seoo-steroids and the normal A -S-ketosteroid substrates for the enzyme. 

A representative example of how enzymes stabilise reactive intermediates is the loss of a proton from the a-position of a carbonyl compound, a rate-limiting step in carbon-carbon bond formations and racemisations, which proceeds via an unstable enol or enolate intermediate. A particularly interesting illustrative case is that of triose-phosphate isomerase (TM) and how it catalyses the isomerisation between dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP) (Figure 14.11). The uphill direction from DHAP to GAP is essential for optimal throughput in the glycolytic pathway, since only GAP can proceed further along this pathway. Thus, the TIM reaction ensures efficient... 

Citrate synthase catalyzes the metabolically important formation of citrate from ace-tyl-CoA and oxaloacetate [68]. Asp-375 (numbering for pig CS) has been shown to be the base for the rate-limiting deprotonation of acetyl-CoA (Fig. 5) [69]. An intennediate (which subsequently attacks the second substrate, oxaloacetate) is believed to be formed in this step the intermediate is thought to be stabilized by a hydrogen bond with His-274. It is uncertain from the experimental data whether this intermediate is the enolate or enol of acetyl-CoA related questions arise in several similar enzymatic reactions such as that catalyzed by triosephosphate isomerase. From the relative pK values of Asp-375... 

Dihydroxyacetone phosphate (82) is a substrate for a-glycero-phosphate dehydrogenase, aldolase, and triose phosphate isomerase, and its O-alkyl ethers are intermediates in the biosynthesis of phospholipids. In neutral aqueous solution at 20 °C, dihydroxyacetone phosphate exists as an equilibrium mixture of the keto (82), gem-d o (83), and enol (84) forms, as shown by n.m.r. spectroscopy. The proportion of (82) to (83)... 

Triose phosphate isomerase is one of the enzymes of glycolysis (see Section 15.2) and is responsible for converting dihydroxyacetone phosphate into glyceraldehyde 3-phosphate by a two-stage enolization process. An intermediate enediol is involved - this common enol can revert to a keto form in two ways, thus providing the means of isomerization. 

We have seen many examples of chemical reactions involving enolate anions, and should now realize just how versatile they are in chemical synthesis (see Chapter 10). We have also seen several examples of how equivalent reactions are utilized in nature. For the triose phosphate isomerase mechanism above, we did not actually invoke a distinct enolate anion intermediate in the enolization process, but proposed that there was a smooth flow of electrons. For other reactions, we shall also need to consider whether enolate anions are actually involved, or whether a more favourable alternative exists. The aldol-type reaction... 

Oxalocrotonate tautomerase. This bacterial enzyme, which functions in the degradation of toluene (Chapter 25), is actually an isomerase. It catalyzes rapid interconversion of an unconjugated unsaturated a-oxoacid such as 4-oxalocrotonate with an intermediate enol (which may leave the enzyme) and the isomeric conjugated oxoacid (Eq. 13-31).168-170 A related 5-carboxymethyl-2-hydroxymuconate isomerase... 

Enzyme mechanisms can often avoid high-energy, unstable cationic or anionic intermediates that increase the reaction barrier. Triosephosphate isomerase catalyzes the tautomerization of the achiral dihydroxy acetone phosphate (DHAP) to f -glyceraldehyde-3-phosphate (G3P) by the mechanism shown below. Not only does the push-pull mechanism avoid forming a highly basic enolate, but the binding mode of DHAP determines which face of the enediol will be protonated in the second step, and therefore... 

Because dihydroxyacetone phosphate and glyceraldehyde 3-phosphate enolize to give a common intermediate, they exist in equilibrium. The enzyme triose phosphate isomerase efficiently catalyzes the isomerization. Although the enediol intermediate is chiral, the enzyme forms only the i enantiomer of glyceraldehyde 3 phosphate. In aqueous solution, an acid-catalyzed reaction would yield a racemic mixture of aldehyde 3-phosphate. 

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