Isomerases 1,2-proton transfer reactions

As first espoused by Knowles and Albery, the limiting selective pressure on enzymatic function is the diffusion-controlled limit by which substrates bind and products dissociate [7]. In the case of triose phosphate isomerase [8], ketosteroid iso-merase [9], mandelate racemase [10], and proline racemase [11], the energies of various transition states on the reactions coordinates have been quantitated, with the result that the free energies of the transition states for the proton transfer reactions to and from carbon are competitive with those for substrate association/ product dissociation. However, as discussed in later sections, the energies of the... 

Isomerases and pyridoxal phosphate (PLP)-dependent transaminases (aminotransferases). The proton transfer reactions associated with isomerases and PLP-dependent transaminases are generally suprafacial processes. This may reflect a mechanistic advantage of a single active site base functioning in both proton abstraction and readdition at the same diastereotopic face of enediol(ate), dienol(ate), or enamine intermediates. 

The aldose-ketose isomerases constitute the best studied class of enzymes catalyzing 1,2-proton transfer reactions (Tables IV and V). Isomerization generally involves significant intramolecular hydrogen transfer with variable amounts of solvent proton exchange (16). This argues for the formation of an enediol(ate) intermediate facilitated by a single active site base partially shielded from solvent [Eq. (16)]. 

Glucose-phosphate isomerase is one of the best studied enzymes catalyzing the interconversion of aldo- and ketohexose phosphates. An active site carboxyl group is a possible candidate for the base catalyzing the intramolecular proton transfer reaction. The affinity label 1,2-anhydro-D-mannitol 6-phosphate (8) inactivates the enzyme by forming an ester linkage between C-l of the affinity label and an active site carboxyl of a glutamic acid residue (98). 

The distribution of 0 between the hydroxyl and hydroxymethylene functions of 19a and 19b, determined by the fragmentation pattern of these two species during mass spectrometry, could only result from addition of the carboxyl of the enzyme to the a face of the steroid. Had the carboxyl added to the spiro carbon from the jS face, the 0 would have been located in the hydroxymethylene function of 19a. Clearly, if the binding of the oxiranyl steroids is analogous to that of steroid substrates, Asp-38 could not be involved in the normal intramolecular proton transfer associated with the isomerization reaction. However, as recently emphasized by Pollack ei al., the oxiranyl steroids could conceivably bind to the active site in an upsidedown orientation in comparison to that of the substrate steroids (133). In this case, Asp-38 would still be the most probable base involved in the intramolecular proton transfer reaction. Perhaps upsidedown binding accounts for the report that the C-4a hydrogen of 5-androstene-3,17-dione undergoes slow labilization in the presence of the isomerase (134, 135). 

Cui Q, M Karplus (2002) Quantum mechanics/molecular mechanics studies of triosephosphate isomerase-catalyzed reactions Effect of geometry and tunneling on proton-transfer rate constants. J. Am. Chem. Soc. 124 (12) 3093-3124... 

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... 

The interiors of proteins are more densely packed than liquids [181], and so the participation of the atoms of the protein surrounding the reactive system in an enzyme-catalysed reaction is likely to be at least as important as for a reaction in solution. There is experimental evidence which indicates that protein dynamics may modulate barriers to reaction in enzymes [10,11]. Ultimately, therefore, the effects of the dynamics of the bulk protein and solvent should be included in calculations on enzyme-catalysed reactions. Dynamic effects in enzyme reactions have been studied in empirical valence bond simulations Neria and Karplus [180] calculated a transmission coefficient of 0.4 for proton transfer in triosephosphate isomerase, a value fairly close to unity, and representing a small dynamical correction. Warshel has argued, based on EVB simulations of reactions in enzymes and in solution, that dynamical effects are similar in both, and therefore that they do not contribute to catalysis [39]. 

The glycolytic pathway includes three such reactions glucose 6-phosphate isomer-ase (1,2-proton transfer), triose phosphate isomerase (1,2-proton transfer), and eno-lase (yS-elimination/dehydration). The tricarboxylic acid cycle includes four citrate synthase (Claisen condensation), aconitase (j5-elimination/dehydration followed by yS-addition/hydration), succinate dehydrogenase (hydride transfer initiated by a-proton abstraction), and fumarase (j5-elimination/dehydration). Many more reactions are found in diverse catabolic and anabolic pathways. Some enzyme-catalyzed proton abstraction reactions are facilitated by organic cofactors, e.g., pyridoxal phosphate-dependent enzymes such as amino acid racemases and transaminases and flavin cofactor-dependent enzymes such as acyl-C-A dehydrogenases others. 

The importance of optimal distance for proton transfer has been emphasized by work on triose-phosphate isomerase. An essential base, Glu-16S, has been replaced by Asp, effectively increasing the bond distance for proton transfer by 1 A (50). The rates of the enzyme-catalyzed enolization steps are reduced 1000-fold (50) relative to wild type. Although the mutant is impaired, its activity is still substantial considering that the wild-type enzyme accelerates the reaction 10 -fold relative to acetate ion in solution. Attempts to select for second-site revertants which restore catalytic activity have met with only modest success (51, 52), but they begin to address the important questions pertaining to the evolution of the optimal geometry of the constellation of amino acids around the active site. 

The kinetics of the reaction of heptene with H2SO4 at various concentrations has been elucidated by UV spectrometry. The efficiency of proton transfer to carbon on the intramolecular ring closing of enol ethers in Kirby s enzyme model for aldolase and isomerase has been investigated computationally by using ab initio and DFT calculation methods... 

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... 

Finally, the use of isotopes in carbon-acid substrates is an invaluable tool for the determination of the stereochemistry of the enzymatic proton transfer. In contrast to organic reactions, stereospecific proton transfers are the rule, rather than the exception, in enzymatic reactions, owing to the inherently asymmetric nature of the protein surface. An example is the pair of isotopic exchange reactions between dihydroxyacetone phosphate and tritiated water catalysed by the enzymes aldolase and triose phosphate isomerase [17]. In the two cases a different a-hydrogen of the ketone is exchanged with water, leading to the two discrete monotritiated derivatives 1 (labelled by the isomerase) and 2 (labelled by the aldolase) ... 

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... 

As will be discussed in Chapter 16, the catalysis of a reaction by aldose-ketose isomerases involves an enediol intermediate in which the transferred proton (T in equation 8.18) remains on the same face of the intermediate. The stereochemistry of the products shows that the intermediate is syn rather than anti.9... 

In the case of the xylose isomerase reaction, for which solvent proton exchange has not been detected, an intramolecular hydride transfer mechanism cannot be excluded (91). 

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