PLP-dependent Racemases

Alanine Racemase (EC 5.1.1.1) Alanine racemase is a PLP-dependent bacterial enzyme that catalyzes the racemization of L- and D-alanine exdusively (for a review see [60]). The enzyme from Salmonella typhimurium is an exception since it also accepts L-Ser, L-homo-Ser, and L-Cys as substrates. Alanine racemase, the most studied member of the PLP-dependent racemases, plays a major role in the bacterial growth by providing D-Ala for the peptidoglycan assembly and cross-Unking. 

Alanine racemase was purified and cloned form various sources. Interestingly, two distinct genes were identified in a number of genome sequences, for example, E. coli, BadUus suhtilis. Pseudomonas aeru nosa, and so on. 

Alanine racemases are homodimeric enzymes of an apparent molecular mass of 76kDa, containing two molecules of PLP as co-enzyme [61] the known enzymes from different sources are highly homologous. With regard to the kinetic properties, the values for D- and L-Ala determined at 30 °C for the Pseudomonas fiuorescens alanine racemase are -12 and 19mM, respectively and the Vnm values for racemization are -1200 and 2200 units/mg protein, respectively. The thermophilic alanine racemase from B. stearothermophilus is quite stable to heat treatment (up to 75 °C for 1 h) while the mesophilic one from B. subtilis is stable up to 55 °C under the same conditions. 

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). 

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. 

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AlaR, the bacterial enzyme that catalyzes the racemization of l- and D-alanine, is the most investigated PLP-dependent amino acid racemase. It is also the only PLP-dependent racemase whose structure has been solved. 

The reversal of this process could potentially occur with reprotonation from either face of the C=N double bond, and a mixture of aldimines would result, leading to generation of a racemic amino acid. This accounts for the mode of action of PLP-dependent amino acid racemase enzymes. Of course, the enzyme controls removal and supply of protons this is not a random event. One important example of this reaction is alanine racemase, employed by bacteria to convert L-alanine into o-alanine for cell-wall synthesis (see Box 13.12). 

PLP-dependent enzymes catalyze the following types of reactions (1) loss of the ce-hydrogen as a proton, resulting in racemization (example alanine racemase), cyclization (example aminocyclopropane carboxylate synthase), or j8-elimation/replacement (example serine dehydratase) (2) loss of the a-carboxylate as carbon dioxide (example glutamate decarboxylase) (3) removal/replacement of a group by aldol cleavage (example threonine aldolase and (4) action via ketimine intermediates (example selenocysteine lyase). 

Alanine racemase, as another PLP-dependent enzyme, is a bacterial enzyme used to create D-alanine from L-alanine for incorporation into the bacterial cell wall. Its role is to act as an electron sink to stabilize carbanionic intermediates generated in enzymatic catalysis. 

In PLP-dependent enzymatic reactions, the Schiff base formed by reaction of the substrate with PLP provides an electron sink for stabilization of the negative charge that results from the bond-breaking process required in the reaction (racemization, decarboxylation, aldol reaction, elimination, etc.). The elegant work of Walsh and coworkers provided evidence that, subsequent to Schiff base formation, a common intermediate is formed from several different alanine analogues that are alanine racemase inhibitors. From this they proposed the elimination-Michael addition sequence shown in Figure 14 as the mechanism for inhibition166. 

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). 

Scheme 13.22 Amino acid racemization by (a) PLP-dependent alanine racemase and (b) PLP-independent a-amino acid racemase.

Serine racemase. The nervous system contains a substantial amount of d-serine, which is generated from 1-serine by serine racemase, a PLP-dependent enzyme. Propose a mechanism for this reaction. What is the equilibrium constant for the reaction 1-serine d-serine ... 

Although not a subject of this chapter, Toney and coworkers have quantitated the reaction coordinate of a PLP-dependent L-alanrne racemase [15]. Despite the expectation that the cofactor provides resonance stabilization of the carbanion/enolate anion (quinonoid) intermediate derived by abstraction of the a-proton, the spectroscopic and kinetic analyses for the wild type racemase at steady-state provided no evidence for the intermediate in the reaction catalyzed by the wild type enzyme. Indeed, Toney had previously demonstrated that a kinetically competent quinonoid intermediate accumulates in the impaired R219E mutant [16] Arg 219 is hydrogen-bonded to the pyridine nitrogen of the cofactor. For the wild type racemase, the derived transition state energies for conversion of the bound enantiomers of alanine,... 

Perhaps the best characterized organic cofactor-dependent racemase is alanine racemase, which employs pyridoxal 5 -phosphate (PLP) (Table 7.1). o-alanine is necessary for the synthesis of the peptidoglycan layer of bacterial cell walls in Gram negative and positive bacteria [1]. Alanine racemase is thus a ubiquitous enzyme in bacteria and an excellent drug target [2]. Both its crystal structure and mechanism have been well investigated. PLP reacts with amino acids to produce... 

The presence of o-serine in mammalian brain tissue was first reported in 1989 [33, 34]. It has recently been established that o-serine is employed in the mammalian forebrain as a co-agonist for the N-methyl-o-asparate (NMDA) excitatory amino acid receptor [35, 36]. A PLP-dependent serine racemase has been cloned and purified from mammalian brain, and found to be a homodimer, which has a number of nonessential cofactors that enhance its activity, including Ca +, Mg + and ATP [37-40]. The mouse brain enzyme has also been shown to catalyze elimination from L-serine, to form pyruvate, with an activity comparable to that for racemiza-tion [41]. Interestingly, the first instance of this class of racemase was discovered by Esaki and coworkers in the silkworm, Bombyx mori [42]. o-serine concentration in the blood of B. mori larvae is thought to play a role in metamorphosis. 

The alanine racemase case . Ala-R is a fold-type III PLP-dependent enzyme implicated in the conversion of L-Ala into D-Ala, the amino acid required for the synthesis of prokaryote cell walls. The absence of Ala-R from eukaryotes, and the absolute requirement of D-alanine, makes it an eligible target for the development of selective antibiotics. " At present, most of the AlaR inhibitors are mono-, di-, or trihalogenated structural analogues of D-Ala and act using different mechanism-hased strategies, able to generate both activated... 

Proton transfer to the imine carbon of the achiral intermediate gives equal amounts of both enantiomers of the PLP imine. The equation illustrates the racemization of L-alanine, which is catalyzed by the PLP-dependent enzyme alanine racemase. Because D-alanine is an essential component of bacterial cell walls, there is considerable interest in designing inhibitors of alanine racemase as potential antibacterial drugs. 

Another enzyme-activated inhibitor is the streptomyces antibiotic D-cycloserine (oxamycin), an antitubercular drug that resembles D-alanine in structure. A potent inhibitor of alanine racemase, it also inhibits die non-PLP, ATP-dependent, D-alanyl-D-alanine synthetase which is needed in the biosynthesis of die peptidoglycan of bacterial cell walls. 

The carboxyl group of an amino acid can also activate the a-hydrogen. This may be the basis for an aspartate racemase and other racemases that are not dependent upon PLP.156-158 See also Chapter 13, Section B,4. 

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