Purification and properties of alanine racemase from crayfish Procambarus clarkii

Shibata, Keitaro; Shirasuna, Katsushi; Motegi, Kengo; Kera, Yoshio; Abe, Hiroki; Yamada, Ryohei

doi:10.1016/s0305-0491(00)00228-5

Cited by 45 publications

(48 citation statements)

References 26 publications

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“…The enzymatic activity of rAlr was determined spectrophotometerically using a colorimetric assay (9). The assay mixture contained 100 mM 2-(N-cyclohexylamino) ethanesulfonic acid (CHES) buffer, (pH 9.0), 2 mM L-alanine, 0.2 U of D amino-acid oxidase, 100 U of catalase, 10 μM PLP and rAlr in a final volume of 0.5 ml.…”

Section: Determination Of Enzyme Activity Enzyme Coupled Colorimetricmentioning

confidence: 99%

Biochemical characterization of Alanine racemase- a spore protein produced by Bacillus anthracis

Kanodia¹,

Agarwal²,

Singh³

et al. 2009

BMB Reports

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Alanine racemase catalyzes the interconversion of L-alanine and D-alanine and plays a crucial role in spore germination and cell wall biosynthesis. In this study, alanine racemase produced by Bacillus anthracis was expressed and purified as a monomer in Escherichia coli and the importance of lysine 41 in the cofactor binding octapeptide and tyrosine 270 in catalysis was evaluated. The native enzyme exhibited an apparent Km of 3 mM for L-alanine, and a Vmax of 295 μmoles/min/mg, with the optimum activity occurring at 37 o C and a pH of 8-9. The activity observed in the absence of exogenous pyridoxal 5 -phosphate suggested that the cofactor is bound to the enzyme. Additionally, the UV-visible absorption spectra indicated that the activity was pH independece, of VV-visible absorption spectra suggests that the bound PLP exists as a protonated Schiff's base. Furthermore, the loss of activity observed in the apoenzyme suggested that bound PLP is required for catalysis. Finally, the enzyme followed non-competitive and mixed inhibition kinetics for hydroxylamine and propionate with a Ki of 160 μM and 30 mM, respectively. [BMB reports 2009; 42(1): 47-52]

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Section: Determination Of Enzyme Activity Enzyme Coupled Colorimetricmentioning

confidence: 99%

Biochemical characterization of Alanine racemase- a spore protein produced by Bacillus anthracis

Kanodia¹,

Agarwal²,

Singh³

et al. 2009

BMB Reports

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“…In contrast, the invertebrate enzyme showed activity even in the absence of PLP. [28][29][30][31] On the other hand, the enzyme was strongly inhibited by several inhibitors specific to PLP-dependent enzymes, suggesting the tightly binding of PLP to the enzyme, probably with a covalent bond as is shown in alanine racemase of Bacillus stearothermophilus, where PLP is covalently linked via an aldimine linkage to lysine 39.…”

Section: Penaeus Monodonmentioning

confidence: 99%

“…Apparent K m values for both L to D and D to L directions were over 160 mM which is typical for crustacean enzymes at least toward L-alanine. 28,30,31) However, V max and k cat of the prawn enzyme were high and almost the same for both directions. Thus, the catalytic efficiency (k cat /K m ) was also not different for both directions.…”

Section: Penaeus Monodonmentioning

confidence: 99%

“…7) However, alanine racemase [EC 5.1.1.1], catalyzing the interconversion between D-and L-alanine, has been detected both in bivalves 5,24) and crustaceans. 25,26) Eukaryotic alanine racemase has recently been purified to homogeneity from a fungus Tolypocladium niveum, 27) crayfish muscle, 28) mantle muscle of C. japonica, 29) hepatopancreas 30) and muscle 31) of black tiger prawn (Table 2). We purified the enzyme to homogeneity from the muscle of Penaeus monodon.…”

Section: D-alanine Synthesis In Aquatic Inverte-bratesmentioning

confidence: 99%

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Physiological Function and Metabolism of Free D-Alanine in Aquatic Animals

Abe

Yoshikawa

Sarower

et al. 2005

Biological & Pharmaceutical Bulletin

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“…Asp-specific racemase has been cloned from microorganisms and shellfish. [43][44][45][46][47][48] However, detailed investigations with mammalian cells or tissues are limited possibly due to extremely low Asp racemase activity or unknown optimal assay conditions. Alternatively, D-Asp may be produced by a different mechanism(s), specifically, via degradation of proteins that contain D-Asp residues and subsequent release of free D-Asp.…”

Section: Adrenal Glandmentioning

confidence: 99%