KINETIC BEHAVIOUR OF CALF-INTESTINAL ALKALINE PHOSPHATASE WITH 4-METHYLUMBELLIFERYL PHOSPHATE
KINETIC BEHAVIOUR OF CALF-INTESTINAL ALKALINE PHOSPHATASE WITH 4-METHYLUMBELLIFERYL PHOSPHATE
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DOI：
10.1042/bj0970095
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发表时间：
1965-01-01
影响因子：
4.1
通讯作者：
WALKER, PG
中科院分区：
文献类型：
--
作者：
FERNLEY, HN;WALKER, PG
测定了不同 pH、离子强度和温度对小牛肠粘膜纯化碱性磷酸酶参数 Km 和 Vmax 的影响，该酶采用新的荧光底物、4-甲基伞形基磷酸单酯二钠盐和氨二醇-盐酸缓冲系统。研究发现，在不同条件下，Km 和 Vmax 之间存在关系，使得 Vmax = β/(1 + α/Km)，其中 a 和 β 是常数，与温度和离子强度相关，但与 pH 无关。结果表明，这种关系令人满意地解释了众所周知的不同底物浓度对最佳 pH 值和速度的影响。各种结果根据 2 种酶形式 E1 和 E2 之间的 pH 依赖性构象平衡进行解释。只有E1与底物结合，只有E2反应生成无机磷酸盐。为了根据该理论解释 Km 和 Vmax 的 pH 变化，假设构象变化与酶中 2 个碱性基团的 pK 变化相关。
The effects of varying pH, ionic strength and temperature on the parameters Km and Vmax for a purified alkaline phosphatase from calf intestinal mucosa with a new fluorogenic substrate, 4-methylumbelliferyl phosphate monoester disodium salt, and an ammediol-hydrochloric acid buffer system were determined. It was found that, under varying conditions, a relationship exists between Km and Vmax, such that Vmax =[beta]/(1 + [alpha]/Km), where a and[beta] are constants, temperature- and ionic strength-dependent, but pH-independent. It is shown that this relationship accounts satisfactorily for the well-known effect of varying substrate concentration on optimum pH and velocity. The various results are interpreted in terms of a pH-dependent conformational equilibrium between 2 forms of the enzyme, E1 and E2. Only E1 combines with substrate, and only E2 reacts to give inorganic phosphate. To account for the pH-variation of Km and Vmax in terms of this theory, it is postulated that the conformational change is associated with a change in pK of 2 basic groups in the enzyme.