Two-base mechanism

The reaction mechanism for glutamate racemase has been studied extensively. It has been proposed that the key for the racemization activity is that the two cysteine residues of the enzyme are located on both sides of the substrate bound to the active site. Thus, one cysteine residue abstracts the a-proton from the substrate, while the other detivers a proton from the opposite side of the intermediate enolate of the amino acid. In this way, the racemase catalyzes the racemization of glutamic acid via a so-called two-base mechanism (Fig. 15). 

The racemization is apparently very simple, but the detailed mechanism remains unsolved. The reaction proceeds via either a one-base or a two-base mechainsm.30,313 In the one-base mechanism, an amino acid residue of the enzyme abstracts the substrate a-proton of the external Schiff base to form an anionic intermediate. The racemization results from the sterically random return of hydrogen to the a-carbon of the intermediate. In the two-base mechanism, two enantiomer-specific bases juxtaposed on either side of the chiral carbon exist in the active center. One base abstracts the a-proton from the external Schiff base, and the conjugated acid of the second base catalyzes protonation to the anionic intermediate from the other side. These roles are reversed for the racemization of the antipodal substrate. 

The one-base mechanism is characterized by the retention of the substrate-derived proton in the product (internal retum).30) With this criterion, reactions catalyzed by a-amino-c-caprolactam racemase,323 amino acid racemase of broad specificity from Pseudomonas striata333 have been considered to proceed through the one-base mechanism. However, such internal returns were not observed in the reactions of alanine racemases from K coli B,33) B. stearothermophilus,263 and S. typhirmaium (DadB and /1/r).263 The internal return should not be observed in the two-base mechanism, because the base catalyzing the protonation to the intermediate probably obtains the proton from the solvent. But the failure of the observation of the internal return can be also explained by the single-base mechanism in which exchange of the proton abstracted from the substrate a-carbon with the solvent is much faster than its transfer to the a-carbanion. Therefore, lack of the internal return does not directly indicate the two-base mechanism of the alanine racemase reaction. 

There is at present no conclusive evidence for either the two-base mechanism or the single-base mechanism. This will probably be shown by a combination of X-ray crystallography and site-directed mutagenesis study, which showed the mechanism of mandelate racemase reactions.42-441... 

Scheme VIII. Proposed two-base mechanism for amino acid racemization by PLP-dependent enzyme (according to [55]).

In attempting to interpret these results one can think in terms of a single base mechanism (Scheme XVIII A), the same base abstracts H-a and protonates C-/3 as well as C-a or C-4, or the involvement of two base groups in these proton transfers. The single base mechanism is rendered very unlikely by the results, because only 1 % of tritium from C-a of the substrate is transferred to the product (i.e., no more than 1% of H-a could be recycled to C-/5) whereas 17% appear at C-4, which must be protonated after C-/5. Thus a two-base mechanism is indicated. An attractive version, shown in Scheme XVIII B, has two base groups situated on opposite faces... 

Scheme XVIII. PLP-substrate Schiff s base geometry for a single-base mechanism (A) and possible geometry for a two-base mechanism (B) for aspartate-/ -decarboxylase.

From these results, the deduced model is that of a two-base mechanism (Figure 12.12). In this model, when a D-isomer of a 5-monosubstituted hydantoin is... 

Figure 12.12 Proposed racemization mechanism of hydantoin racemase using a two-base mechanism. The hypothetical character of the intermediate structure is indicated by the dashed lines surrounding the figure.

A variety of amino acid racemases have been identified in bacteria, archaea, and eukaryotes. They are dassified into two groups pyridoxal 5 -phosphate (PLP) -dependent and -independent enzymes. Therefore, racemization can be achieved via two mechanisms through a chiraUy unstable Schiff base intermediate with an aromatic aldehyde serving as co-factor PLP (Scheme 13.22a) and by a two-base mechanism without co-factor (Scheme 13.22b). 

The members of this dass follow a two-base mechanism involving two cysteine residues as the conjugated catalytic add and base for the abstraction of the a-H from both amino adds. 

Figure 7.2. A one-base versus a two-base mechanism for deprotonation/reprotonation with a planar carbanionic intermediate. In a one-base mechanism, either the catalytic base...

Table 7.2. Permutations of the two-base mechanism for racemization/epimerization.

In addition to n-alanine and n-glutamate, many bacterial cell walls also contain meso-diaminopimelate (DAP) [2]. DAP is produced by epimerization from l,l-DAP to d,l-DAP by the cofactor independent diaminopimelate epimerase [97, 98]. The structure of this enzyme has been solved and two cysteines in the active site were proposed to be the acid-base catalysts [99]. The pattern of label incorporation from tritiated water is consistent with a two-base mechanism [97]. The enzyme has been shown to be stoichiometrically inhibited by the thiol alkylating agent aziDAP [97]. Interestingly, DAP epimerase has an equilibrium constant of 2 (Keq = [d,l]/[l,l]) duc to the statistically expected higher concentration of the [d,l] form at equilibrium between these species [100]. 

There are a number of cofactor independent carbohydrate epimerases that act on activated substrates, such as keto-sugars and keto-sugar nucleotides, although there is a paucity of details about their mechanisms. D-ribulose-5-phosphate 3-epimerase catalyzes the stereoinversion of substrate about the C-3 carbon to form D-xylulose 5-phosphate (as in Fig. 7.15) [102, 103]. Solvent hydron is completely incorporated into the product at the C-3 carbon, during epimerization in the d-xylulose 5-phosphate to o-ribulose 5-phosphate direction [102], This was taken as evidence for a two-base mechanism. 

Epimerases and racemases may or may not employ enzyme cofactors (organic or inorganic) to activate the stereogenic center of the substrate. Common cofactor-stabilized intermediates include resonance-stabilized carbanions and metal-stabilized enolates. The substrate itself can be intrinsically activated if the stereogenic center is adjacent to a carbonyl or carboxylate group. A preponderance of racemases and epimerases act on activated substrates. A number of sugar and sugar nucleotide epimerases act on unactivated substrates. Double proton transfers may proceed, in principle, by either a one- or two-base mechanism. However, only two-base mechanisms have been observed for racemases. 

Structural evidence and site-directed mutagenesis studies strongly support the two-base mechanism involving the phenolate anion of Tyr265 for the deprotonation of the substrate This two-base mechanism... 

The observation of net retention of configuration by the muscle enzyme, as well as the other aldolases, suggests that a single active site base could alternatively function in a general base and general acid capacity in the exchange and condensation reactions, respectively (Fig. 7) (76). However, the stereochemistry does not absolutely require a single-base mechanism. For example, Hupe and co-workers propose a two-base mechanism in which the phosphate at C-1 of the... 

Racemases are enzymes that catalyze the inversion of the chiral center by deprotonation of the C , followed by reprotonation on the opposite face of the planar carban-ionic transition-state species [13,14], In order to overcome the high energetic barrier of racemization, for example, on a-amino acids, some racemases employ pyridoxal phosphate (PLP) as a cofactor to use the resonance-stabilized amino acid complex as an electron sink because the estimated pK values for the C of amino acids are high, in the range 21-32 [14,15]. The formation of an imine PLP-substrate covalent bond makes the pK value of a-hydrogen of amino acids low. The second class of enzymes includes proline, aspartate, and glutamate racemases and diaminopimelate epimer-ase, with a cofactor-independent two-base mechanism [14],... 

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