ALBUMINS ACTIVATE PEPTIDE HYDROLYSIS BY THE BIFUNCTIONAL ENZYME LTA4 HYDROLASE AMINOPEPTIDASE
ALBUMINS ACTIVATE PEPTIDE HYDROLYSIS BY THE BIFUNCTIONAL ENZYME LTA4 HYDROLASE AMINOPEPTIDASE
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DOI：
10.1021/bi00132a010
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发表时间：
1992-05-05
期刊：
影响因子：
2.9
通讯作者：
FITZPATRICK, FA
中科院分区：
文献类型：
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作者：
ORNING, L;FITZPATRICK, FA
来自几个物种的白蛋白激活双功能Zn 2+金属酶氨肽酶/白三烯A4水解酶（EC 3.3.2.6）。牛血清白蛋白（1 mg/mL）分别使L-脯氨酸-对硝基苯胺和亮氨酸-脑啡肽的水解增加12倍和7倍。L-脯氨酸-对硝基苯胺的表观K（m）与白蛋白浓度成反比，从0至1 mg/mL，从9.4降至0.7 mM，表观V（max）无明显变化。这些数据意味着一个随机的激活过程中，酶激活剂复合物是催化占主导地位。Hill图表明白蛋白和酶之间的1：1化学计量关系。斜率与白蛋白浓度倒数的二次曲线表明，它与酶结合的亲和力常数为0.9 μ M。非活化酶的最适pH值发生在pH 8;白蛋白活化酶的最适pH值接近7。无论是超滤或透析白蛋白改变其激活作用，但煮沸取消it. Albumin不影响其他胞质或微粒体亮氨酸氨基肽酶，或γ-谷氨酰转移酶。白蛋白作为一种非必需的激活剂发挥作用，因为在其不存在的情况下总是可检测到酶活性。氯离子可激活其他Zn 2+金属酶，也可激活白三烯A4水解酶/氨肽酶，EC 50 = 50 mM，在不存在白蛋白的情况下使其初速度增加2.2倍。Zn ~（2+）激活酶，增加其表观V（max），但不增加其表观K（m），表明它取代了从活性位点丢失的Zn ~（2+），特别是在酸性pH下。白蛋白减轻氯化物的影响，但没有锌2+或竞争性抑制剂，卡托普利的影响。其他几种蛋白质，包括明胶，牛或人γ-球蛋白，人α-2-巨球蛋白，和牛甲胎蛋白没有刺激LTA 4水解酶的氨肽酶活性。我们的研究结果表明，LTA 4水解酶/氨肽酶和白蛋白之间的相互作用引起的酶的构象变化，从而增加肽的亲和力。
Albumins from several species activated the bifunctional, Zn2+ metalloenzyme aminopeptidase/leukotriene A4 hydrolase (EC 3.3.2.6). Bovine serum albumin, 1 mg/mL, increased hydrolysis of L-proline-p-nitroanilide and leucine-enkephalin by 12-fold and 7-fold, respectively. The apparent K(m) for L-proline-p-nitroanilide was inversely proportional to the albumin concentration from 0 to 1 mg/mL, declining from 9.4 to 0.7 mM without an appreciable change in apparent V(max). These data imply a random activation process in which the enzyme-activator complex is catalytically dominant. Hill plots indicated a 1:1 stoichiometric relationship between albumin and enzyme. Secondary plots of slope versus the reciprocal of albumin concentration indicated that it binds to the enzyme with an affinity constant of 0.9-mu-M. The pH optimum of the nonactivated enzyme occurred at pH 8; the albumin-activated enzyme had an optimum near pH 7. Neither ultrafiltration nor dialysis of albumin altered its activating effect, but boiling abolished it. Albumin did not affect other cytosolic or microsomal leucine aminopeptidases, or gamma-glutamyltransferase. Albumin functions as a nonessential activator, since enzymatic activity was always detectable in its absence. Chloride ions, which activate other Zn2+ metalloenzymes, also activated leukotriene A4 hydrolase/aminopeptidase with an EC50 = 50 mM, increasing its initial velocity 2.2-fold in the absence of albumin. Zn2+ activated the enzyme, increasing its apparent V(max) but not its apparent K(m), suggesting it replaced Zn2+ lost from the active site, especially at acidic pH. At concentrations greater than 30-50-mu-M, Zn2+ was inhibitory. Albumin mitigated the effect of chloride, but not the effect of Zn2+ or that of the competitive inhibitor, captopril. Several other proteins including gelatin, bovine or human gamma-globulins, human alpha-2-macroglobulin, and bovine alpha-fetoprotein did not stimulate the aminopeptidase activity of LTA4 hydrolase. Our results suggest that an interaction between LTA4 hydrolase/aminopeptidase and albumin causes a conformational change in the enzyme, leading to an increased affinity for peptides.