Reaction Mechanism of Alanine Racemase

The alanine racemization catalyzed by alanine racemase is considered to be initiated by the transaldimination (Fig. 8.5).26) In this step, PLP bound to the active-site lysine residue forms the external Schiff base with a substrate alanine (Fig. 8.5, 1). The following a-proton abstraction produces the resonance-stabilized carbanion intermediates (Fig. 8.5, 2). If the reprotonation occurs on the opposite face of the substrate-PLP complex on which the proton-abstraction proceeds, the antipodal aldimine is formed (Fig. 8.5,3). The subsequent hydrolysis of the aldimine complex gives the isomerized alanine and PLP-form racemase. The random return of hydrogen to the carbanion intermediate is the distinguishing feature that differentiates racemization from reactions catalyzed by other pyridoxal enzymes such as transaminases. Transaminases catalyze the transfer of amino group between amino acid and keto acid, and the reaction is initiated by the transaldimination, followed by the a-proton abstraction from the substrate-PLP aldimine to form a resonance-stabilized carbanion. This step is common to racemases and transaminases. However, in the transamination the abstracted proton is then tranferred to C4 carbon of PLP in a highly stereospecific manner The re-protonation occurs on the same face of the PLP-substrate aldimine on which the a-proton is abstracted. With only a few exceptions,27,28) each step of pyridoxal enzymes-catalyzed reaction proceeds on only one side of the planar PLP-substrate complex. However, in the amino acid racemase 

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. 

Faraci and Walsh263 studied the substrate and solvent deuterium isotope effects of the reactions catalyzed by alanine racemases of S. typhimurium (DadB and Air enzymes) and B. stearothermophilus. Although the kinetic constants for all three alanine racemases obey the Haldane equation, i.e., Keq= 1 (this confirms that the enzymes are true racemases), the individual Micaelis-Menten parameters in both directions show marked difference in the binding of each isomer. This suggests a structural asymmetry at the active sites of these enzymes. The asymmetry in the recognition and turnover of substrate enantiomer was also clearly seen in the results of isotope effect experiment with DadB enzyme. In the d- 

---

Fig. 8.5 Reaction mechanism of alanine racemase. (Reproduced with permission from Faraci and Walsh, Biochemistry, 27, 3268 (1988)).

---