首页> 美国卫生研究院文献>Biochemical Journal >The catalytic consequences of experimental evolution. Studies on the subunit structure of the second (ebg) beta-galactosidase of Escherichia coli and on catalysis by ebgab an experimental evolvant containing two amino acid substitutions.
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The catalytic consequences of experimental evolution. Studies on the subunit structure of the second (ebg) beta-galactosidase of Escherichia coli and on catalysis by ebgab an experimental evolvant containing two amino acid substitutions.

机译:实验进化的催化结果。研究大肠杆菌第二个(ebg)β-半乳糖苷酶的亚基结构以及由ebgab催化的实验ebgab是一种包含两个氨基酸取代的实验进化产物。

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摘要

1. The ratio of ebgA-gene product of ebgC-gene product in the functional aggregate of ebg beta-galactosidases was determined to be 1:1 by isolation of the enzyme from bacteria grown on uniformly radiolabelled amino acids and separation of the subunits by gel-permeation chromatography under denaturing conditions. 2. This datum, taken together with a recalculation of the previous ultracentrifuge data [Hall (1976) J. Mol. Biol. 107, 71-84], analytical gel-permeation chromatography and electron microscopy, strongly suggests an alpha 4 beta 4 quaternary structure for the enzyme. 3. The second chemical step in the enzyme turnover sequence, hydrolysis of the galactosyl-enzyme intermediate, is markedly slower for ebgab, having both Asp-97----Asn and Trp-977----Cys changes in the large subunit, than for ebga (having only the first change) and ebgb (having only the second), and is so slow as to be rate-determining even for an S-glycoside, beta-D-galactopyranosyl thiopicrate, as is shown by nucleophilic competition with methanol. 4. The selectivity of galactosyl-ebgab between water and methanol on a molar basis is 57, similar to the value for galactosyl-ebgb. 5. The equilibrium constant for the hydrolysis of lactose at 37 degrees C is 152 +/- 19 M, that for hydrolysis of allolactose is approx. 44 M and that for hydrolysis of lactulose is approx. 40 M. 6. A comparison of the free-energy profiles for the hydrolyses of lactose catalysed by the double mutant with those for the wild-type and the single mutants reveals that free-energy changes from the two mutations are not in general independently additive, but that the changes generally are in the direction predicted by the theory of Burbaum, Raines, Albery & Knowles [(1989) Biochemistry 28, 9283-9305] for an enzyme catalysing a thermodynamically irreversible reaction. 7. Michaelis-Menten parameters for the hydrolysis of six beta-D-galactopyranosylpyridinium ions and ten aryl beta-galactosides by ebgab were measured. 8. The derived beta 1g values are the same as those for ebgb (which has only the Trp-977----Cys change) and significantly different from those for ebgo (the wild-type enzyme) and ebga. 9. The alpha- and beta-deuterium secondary isotope effects on the hydrolysis of the galactosyl-enzyme of 1.08 and 1.00 are difficult to reconcile with the pyranose ring in this intermediate being in the 4C1 conformation.
机译:1.通过从生长在放射性同位素标记的氨基酸上的细菌中分离出酶,并通过凝胶分离亚基,确定ebgβ-半乳糖苷酶功能聚集体中ebgC基因产物的ebgA基因产物之比为1:1。 -变性条件下的渗透色谱。 2.该数据与先前超速离心机数据的重新计算一起使用[Hall(1976)J. Mol。Chem。生物学[107,71-84],分析性凝胶渗透色谱法和电子显微镜,强烈暗示了该酶的α4β4季结构。 3.酶转换序列中的第二个化学步骤,即半乳糖基酶中间体的水解,对于依巴单抗而言明显较慢,因为大亚基中具有Asp-97 ---- Asn和Trp-977 ---- Cys变化,比ebga(仅具有第一个变化)和ebgb(仅具有第二个变化)要慢,甚至对于S-糖苷,β-D-吡喃半乳糖基硫代庚酸酯而言,其速率也很慢,如亲核竞争所示用甲醇。 4.水和甲醇之间的半乳糖基-依巴格比的选择性为57,与半乳糖基-依巴格比的值相似。 5.在37℃下水解乳糖的平衡常数为152 +/- 19M,而对半乳糖水解的平衡常数为约3。大约44 M,而乳果糖水解大约为44M。 40 M. 6.比较双突变体与野生型和单突变体催化的乳糖水解的自由能谱表明,两个突变体的自由能变化通常不会独立累加但是这种变化通常是在Burbaum,Raines,Albery&Knowles [(1989)Biochemistry 28,9283-9305]的理论上预测的,该酶催化热力学不可逆的反应。 7.测量了依贝加布水解六个β-D-吡喃半乳糖基吡啶鎓离子和十个芳基β-半乳糖苷的Michaelis-Menten参数。 8.得出的beta 1g值与ebgb(仅具有Trp-977 ---- Cys变化)的值相同,并且与ebgo(野生型酶)和ebga的值显着不同。 9.α-和β-氘的二次同位素对1.08和1.00的半乳糖基酶的水解作用难以与该中间体呈4C1构型的吡喃糖环调和。

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