反硝化微球菌中赤藓糖-β-羟基天冬氨酸脱水酶的纯化及性质

Purification and properties of erythro-beta-hydroxyasparate dehydratase from Micrococcus denitrificans.

作者信息

Gibbs R G, Morris J G

机构信息

Department of Biochemistry, University of Leicester.

出版信息

Biochem J. 1965 Nov;97(2):547-54. doi: 10.1042/bj0970547.

DOI:10.1042/bj0970547

PMID:16749162

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1264673/

Abstract

The novel enzyme, erythro-beta-hydroxyaspartate dehydratase, a key enzyme of the beta-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-beta-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-beta-hydroxyaspartate. 3. The enzyme was highly substrate-specific, utilizing only the l-isomer of erythro-beta-hydroxyaspartate (K(m), 0.43mm, and V(max.), 99mumoles of oxaloacetate formed/min./mg. of protein at pH9.15 and 30 degrees ). Of many compounds tested, only maleate was a competitive inhibitor (K(i), 32mm at pH7.6). 4. The optimum pH for activity was about 9.5. The K(m) varied with pH, showing a marked optimum at pH7.8. The V(max.) 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 (Mg(2+), Ca(2+) and Mn(2+)); no activation by univalent cations was observed. 8. The findings are discussed in the light of reported properties of related hydroxyamino acid dehydratases.

摘要

新型酶——赤藓糖-β-羟基天冬氨酸脱水酶，是β-羟基天冬氨酸途径的关键酶（科恩伯格和莫里斯，1963年，1965年），已从以乙醇酸盐培养的反硝化微球菌提取物中纯化了30倍。纯化后的制剂缺乏赤藓糖-β-羟基天冬氨酸醛缩酶活性，且不含作用于草酰乙酸的酶。2. 通过直接测定添加的赤藓糖-β-羟基天冬氨酸酶促形成草酰乙酸和氨的方法，研究了纯化脱水酶的性质。3. 该酶具有高度的底物特异性，仅利用赤藓糖-β-羟基天冬氨酸的L-异构体（在pH9.15和30℃时，米氏常数（K(m)）为0.43mmol，最大反应速度（V(max.)）为每分钟每毫克蛋白质形成99微摩尔草酰乙酸））。在测试的许多化合物中，只有马来酸盐是竞争性抑制剂（在pH7.6时，抑制常数（K(i)）为32mmol）。4. 活性的最适pH约为9.5。米氏常数随pH变化，在pH7.8时显示出明显的最适值。最大反应速度也随pH变化，表明酶-底物复合物中存在解离常数（pK'(a)）约为8.5的可解离基团。5. 羰基试剂具有抑制作用，但在测试的三种硫醇试剂中，只有对氯汞苯甲酸具有抑制作用。6. 该酶的部分解离制剂通过添加磷酸吡哆醛而被激活了四倍，从而恢复到一半活性。7. 乙二胺四乙酸（EDTA，0.1mmol）几乎完全抑制活性，二价阳离子（Mg(2+)、Ca(2+)和Mn(2+)）可恢复活性；未观察到一价阳离子的激活作用。8. 根据相关羟基氨基酸脱水酶的报道性质对这些发现进行了讨论。