Racemase, active site

Figure 17-19. Models of the mandelate racemase active site with complexed substrate, p-iodomandelate. Reprinted from Neidhartet al.11941.

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). 

Decarboxylases are one of the members of the enolase superfamily. The most important and interesting point of this class of enzymes is that they are mechanistically diverse and catalyze different overall reactions. However, each enzyme shares a partial reaction in which an active site base abstracts a proton to form a nucleophile. The intermediates are directed to different products in the different active sites of different members. However, some enzymes of this class exhibit catalytic promiscuity in their natural form. ° This fact is considered to be strongly related to the evolution of enzymes. Reflecting the similarity of the essential step of the total reaction, there are some successful examples of artificial-directed evolution of these enzymes to catalyze distinctly different chemical transformation. The changing of decarboxylase to racemase described in Section 2.5 is also one of these examples. 

Other Proteins The ouabain-binding site on (Na /K -adenosine-5 -triphosphatase, 46, 523 penicillin isocyanates for /3-lactamase, 46, 531 active site-directed addition of a small group to an enzyme the ethylation of ludferin, 46, 537 mandelate racemase, 46, 541 d imethylpyrazole carboxamidine and related derivatives, 46, 548 labeling of catechol O-methyltransferase with N-haloace-tyl derivatives, 46, 554 affinity labeling of binding sites in proteins by sensitized photooxidation, 46, 561 bromocolchicine as a iabei for tubuiin, 46, 567. 

Very detailed studies on the inhibition of alanine racemase by fluoroalanines have been conducted. This enzyme catalyzes the racemization of alanine to provide D-alanine, which is required for synthesis of the bacterial wall. This work has demonstrated that a more complex process than that represented in Figure 7.47 could intervene. For instance, in the case of monofluoroalanine, a second path (Figure 7.48, path b) occurs lysine-38 of the active site can also attack the Schiff base PLP-aminoacrylate that comes from the elimination of the fluorine atom. This enamine inactivation process (path b) has been confirmed by isolation and identification of the alkylation compound, after denaturation of the enzyme (Figure 7.48). ... 

Figure 13-5 An S-mandelate ion in the active site of mandelate racemase. Only some of the polar groups surrounding the active site are shown. The enzyme has two catalytic acid-base groups. Lysine 166 is thought to deprotonate S-mandelate to form the aci anion, while His 297 deprotonates R-mandelate to form the same anion.106...

P. C. Babbitt, J. A. Gerlt, G. A. Petsko, and D. Ringe, Evolution of an enzyme active site the structure of a new crystal form of muconate lactonizing enzyme compared with mandelate racemase and enolase, Proc. Natl. Acad. Sci. USA 1998,... 

Fig. 8.1 Linear alignment of the protein sequences of alanine racemases from B. subtilis, B. stearothermophilus, S. typhimurium dadB, and S. typhimurium air. The sequences of four alanine racemases were aligned by introducing gaps (hyphens) to maximize identities. Common residues among the four ( ) and three ( ) enzymes are shown below. The active-site lysyl residue is indicated with an asterisk. The vertical arrow shows the position where the limited proteolysis occurs. (Reproduced with permission from Tanizawa el al., Biochemistry, 27, 1311 (1988)).

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... 

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-... 

The racemase enzymes are also of interest in this connection. Both enantiomers function as substrates and presumably bind to the same active site. 

Figure 12.11 Superposition of active sites from the different proteins. The active site of SmeHyuA model (gray) is compared with that of (a) aspartate racemase from Pyrococcus horikoshii (IF)LA, black) and that of (b) glutamate racemase from Aquifex pyrophilus (1B73A, black).

Figure 13.1 Schematic representation of interactions at the active site of (a) R. gracilis D-amino acid oxidase in complex with CFs-D-alanine (pdb ICOL), (b) C. rhodostoma L-amino acid oxidase in complex with citrate (pdb IF8R), and (c) 6. stearothermophilus alanine racemase (pdb ISFT).

Serine Racemase (EC 5.1.1.16] Serine racemases have been discovered in both bacteria and eukaryotes (for a review see [60, 62). In the latter organisms, serine racemase catalyzing the conversion of L-Ser to D-Ser was at first discovered in the silkworm Bombyx mori it is a PLP-dependent racemase which is also active on L-Ala (-6% of the activity on L-Ser). A serine racemase was also purified from rat brain (and a serine racemase cDNA was cloned from mouse brain). Mammalian serine racemase shows sequence simUarily with L-threonine dehydratase from various sources all the active site residues of the latter enzyme are also conserved in mouse serine racemase. Mammalian serine racemase is a member of the fold-type II group of PLP enzymes (similarly to L-threonine dehydratase, D-serine dehydratase, and so on) and distinct from alanine racemase, which belongs to the fold-type III group. Mouse serine racemase shows a low kinetic efficiency the Km values for L- and D-Ser are -10 and 60 mM, respectively and the V ax values with L- and D-Ser are 0.08 and 0.37 units/mg protein (less than 0.1% of those of alanine racemase on L- and D-Ala, see above). 

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