Enzyme aspartate racemase

Aspartate a-decarboxylase 753, 755 Aspartate p-decarboxylase 746 Aspartate racemase 741 Aspartic acid (Asp, D) 52, 53s biosynthesis 517 pXa value of 293, 487 Aspartic proteases 621-625 Aspartyl aminopeptidase 621 p-Aspartyl phosphate 539, 540s Assays of enzyme activity 456 Assembly core of virus shell 365 Assembly pathway... 

The procedure reported in Scheme 13.11 describes deracemization of an amino acid involving oxidation with an L-specific enzyme and transamination with a D-amino transferase using D-aspartate 10, which is generated from L-aspartate 11 by aspartate racemase, as the amino donor. The oxidative enzyme is defined as an L-amino acid deaminase, a flavoprotein from Proteus myxofadens [34]. The transamination reaction is shifted towards the product since the oxalacetate 12 formed decarboxylates spontaneously to give pyruvate and carbon dioxide. 

Okada et al. purified the enzyme to homogeneity from the cell extract of S. thermophilus, the specific activity of the crude extract of which was elevated 3400-fold1106. The gene encoding aspartate racemase was cloned from S. thermophilus, and overexpressed in E. colillll The amount of the enzyme produced reached... 

Yamauchi et al.11121 concluded that aspartate racemase also uses two bases to remove and return the a-proton of the substrate. Aspartate racemase contains three cysteine residues Cys 84, Cys 190 and Cys 197, and only Cys 84 is essential for the enzyme activity. The alkylation of one cysteine residue/dimer with 2-nitro-5-thiocya-nobenzoic acid results in a complete loss of activity. Therefore, the enzyme shows a half-of-the-sites-reactivity11121. Yamauchi et al.11121 suggested that the enzyme has a composite active site formed at the interface of two identical subunits in the same manner as proposed for proline racemase1921. 

After formation of the aldimine, numerous factors in the enzyme facilitate deprotonation of the a-carbon (Fig. 3, Step II). The lysine liberated by transimi-nation is utilized as a general base and is properly oriented for effective deprotonation [11]. Furthermore, the inductive effects of the ring system are tuned to increase the stabilization of the quinoid intermediate. For example, the aspartate group that interacts with the pyridyl nitrogen of the co enzyme promotes proto-nation to allow the ring to act as a more effective electron sink. In contrast, in alanine racemase, a less basic arginine residue in place of the aspartic acid is believed to favor racemization over transamination [12]. 

Wolosker, H., Blackshaw, S., Snyder, S. H. Serine racemase a glial enzyme synthesizing D-serine fo regulate glutamate-N-methyl-D-aspartate neurotransmission, Proc. Natl. Acad. Sci. USA 1999,... 

Other amino acids such as L-aspartate and L-serine, can spontaneously racemize with aging in humans [149]. It has been reported that microwaving milk racemizes L-proline to D-proline, which could reduce the milk s nutritional value [150]. For L-serine, a serine racemase has been characterized that catalyzes the direct racemization of L-serine to D-serine. This enzyme can also catalyse D-serine to L-serine but with lesser affinity. As a number... 

Scheme 12.2. The reversible transamination of aspartate (Asp, D) and alanine (Ala, A) as catalyzed by aminotransferase using pyridoxal as a cofactor. In principle (and apparently in practice), the same process can be used to convert any a-ketocarboxyhc acid to the corresponding amino acid. The a-proton is picked up and deposited on the same side (unless a racemase is involved) of the two amino acids. EC numbers and some graphic materials provided in this scheme have been taken from appropriate hnks in a URL starting with http // www.chem.qmul.ac.uk/iubmb/enzyme/.

The substrate, L-aspartate, is produced from fumarate by an enzyme system involving aspartase, as described in the section on L-aspartate. To produce L-alanine directly from fumarate, the L-alanine-producing column was connected in tandem to an L-aspartate-producing column. In this tandem column system, side reactions caused by fumarase in Escherichia coli and alanine racemase in P. dacunhae reduced the yield. Then, both bacterial cells were separately treated with high temperature and low pH, respectively, and the enzymes responsible for the side reactions were inactivated. Immobilization of these two kinds of bacterial cells with K-carrageenan resulted in the production of L-alanine in a single reactor without the production of the side products, malate and o-alanine (Takamatsu et al. 1982 Chibata et al. 1986b). 

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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