ALKALINE-PHOSPHATASE IS AN ALMOST PERFECT ENZYME
ALKALINE-PHOSPHATASE IS AN ALMOST PERFECT ENZYME
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DOI：
10.1021/bi00200a018
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发表时间：
1994-08-30
期刊：
影响因子：
2.9
通讯作者：
JENCKS, WP
中科院分区：
文献类型：
--
作者：
SIMOPOULOS, TT;JENCKS, WP
在蔗糖或阿拉伯糖存在下，碱性磷酸酶催化4-硝基苯基磷酸酯水解的二级速率常数k（cat）/K-m随粘度增加而降低，在pH 8.0，25 ℃下，偏导数斜率[（k（cat）/K-m）（0）/（k（cat）/K-m）]/偏导数（eta/eta（0））= 1.4。这与底物与活性酶的限速扩散相遇一致，表明碱性磷酸酶是一种“完美的酶”。然而，所报道的二级速率常数k（cat）/K-m = 6.6 X 10（6）至4.6 X 10（7）M（-1）s（-1）小于扩散极限;这表明只有大约0.1-1%的扩散接触是有效的。的一阶速率常数，k（猫），限速水解的磷酸酶中间体在pH 6与饱和底物浓度是独立的蔗糖水溶液中的粘度。这表明蔗糖不会使磷酸酶水解的过渡态不稳定。然而，在pH 8.0下，产物解离是速率限制的，并且在蔗糖存在下k（cat）随着粘度增加而降低，偏导数（k（0）/k（obsd））/偏导数（eta/eta（0））的斜率在0.04 M Mops缓冲液中= 1.2，在0.1 M Tris缓冲液中为1.0，在0.67 M Tris缓冲液中为1.2。这与无机磷酸盐和Tris磷酸盐从酶中的限速扩散分离是一致的。相比之下，甘油在pH 8.0下引起k（cat）/K-m的大幅降低，并且在pH 6下也降低k（cat）。这表明甘油通过溶剂对酶催化活性的影响以及增加粘度来降低速率。
The second-order rate constant, k(cat)/K-m, for catalysis of the hydrolysis of 4-nitrophenyl phosphate by alkaline phosphatase decreases with increasing viscosity in the presence of sucrose or arabinose, with a slope of partial derivative[(k(cat)/K-m)(0)/(k(cat)/K-m)]/partial derivative(eta/eta(0)) = 1.4 at pH 8.0, 25 degrees C. This is consistent with rate-limiting diffusional encounter of the substrate with active enzyme and indicates that alkaline phosphatase is a ''perfect enzyme''. However, the reported second-order rate constants of k(cat)/K-m = 6.6 X 10(6) to 4.6 X 10(7) M(-1) s(-1) are smaller than the diffusional limit; this shows that only similar to 0.1-1% of the diffusional encounters are productive. The first-order rate constant, k(cat), for rate-limiting hydrolysis of the phosphoenzyme intermediate at pH 6 with saturating substrate concentration is independent of viscosity in aqueous sucrose solutions. This shows that sucrose does not destabilize the transition state for phosphoenzyme hydrolysis. However, at pH 8.0 product dissociation is rate limiting and k(cat) decreases with increasing viscosity in the presence of sucrose, with slopes of partial derivative(k(0)/k(obsd))/partial derivative(eta/eta(0)) = 1.2 in 0.04 M Mops buffer, 1.0 in 0.1 M Tris, and 1.2 in 0.67 M Tris buffer. This is consistent with rate-limiting diffusional separation of inorganic phosphate and of Tris phosphate from the enzyme. In contrast, glycerol causes a large decrease in k(cat)/K-m at pH 8.0 and also decreases k(cat) at pH 6. This shows that glycerol decreases the rate by a solvent effect on the catalytic activity of the enzyme, as well as by increasing the viscosity.