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. 1965 Nov;97(2):547–554. doi: 10.1042/bj0970547

Purification and properties of erythro-β-hydroxyasparate dehydratase from Micrococcus denitrificans

R G Gibbs 1, J G Morris 1
PMCID: PMC1264673  PMID: 16749162

Abstract

1. The novel enzyme, erythro-β-hydroxyaspartate dehydratase, a key enzyme of the β-hydroxyaspartate pathway (Kornberg & Morris, 1963, 1965), has been purified 30-fold from extracts of glycollate-grown Micrococcus denitrificans. The purified preparation was devoid of erythro-β-hydroxyaspartate-aldolase activity, and free from enzymes that act on oxaloacetate. 2. Properties of the purified dehydratase were studied by direct assay of the enzymic formation of oxaloacetate and ammonia from added erythro-β-hydroxyaspartate. 3. The enzyme was highly substrate-specific, utilizing only the l-isomer of erythro-β-hydroxyaspartate (Km, 0·43mm, and Vmax., 99μmoles of oxaloacetate formed/min./mg. of protein at pH9·15 and 30°). Of many compounds tested, only maleate was a competitive inhibitor (Ki, 32mm at pH7·6). 4. The optimum pH for activity was about 9·5. The Km varied with pH, showing a marked optimum at pH7·8. The Vmax. also varied with pH in a manner suggesting the presence in the enzyme–substrate complex of a dissociable group of pK′a about 8·5. 5. Carbonyl reagents were inhibitory, but of three thiol reagents tested only p-chloromercuribenzoate was inhibitory. 6. A partially resolved preparation of the enzyme was activated four-fold by the addition of pyridoxal phosphate and thereby restored to half activity. 7. EDTA (0·1mm) was almost completely inhibitory, activity being restored by bivalent cations (Mg2+, Ca2+ and Mn2+); no activation by univalent cations was observed. 8. The findings are discussed in the light of reported properties of related hydroxyamino acid dehydratases.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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