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

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Abstract
The novel enzyme, erythro-fi-hydroxyaspartate dehydratase, a key enzyme of the /3-hydroxyaspartate pathway (Kornberg and Morris, 1963, 1965), has been purified 30-fold from extracts of glycollate-grown M. denitrificans. The purified preparation was devoid of erythro-[beta]-hydroxyaspartate-aldolase activiy, and free from enzymes that act on oxaloacetate. Properties of the purified dehydratase were studied by direct assay of the enzymic formation of oxaloacetate and ammonia from added erythro-[beta]-hydroxyaspartate. The enzyme was highly substrate-specific, utilizing only the L-isomer of erythro-[beta]-hydroxy-aspartate (Km, 0.43 m[image], and Vmax., 99 [mu]moles of oxaloacetate formed/ min./mg of protein at pH 9.15 and 30[degree]). Of many compounds tested, only maleate was a competitive inhibitor (Ki, 32m[image] at pH 7.6). The optimum pH for activity was about 9.5. The Km varied with pH, showing a marked optimum at pH 7.8. 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. Carbonyl reagents were inhibitory, but of three thiol reagents tested only p-chloro-mercuribenzoate was inhibitory. A partially resolved preparation of the enzyme was activated fourfold by the addition of pyridoxal phosphate and thereby restored to half activity. EDTA (0.1 m[image]) was almost completely inhibitory, activity being restored by bivalent cations (Mg2+, Ca2+ and Mn2+); no activation by unlvalent cations was observed. The findings are discussed in the light of reported properties of related hydroxyamino acid dehydratases.