机械的酪氨酸的角色124年149年和丝氨酸UDP-galactose 4-epimerase从大肠杆菌。
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刘Y, Thoden JB,金正日J,伯杰E,活动,拉·FJ,霍尔顿嗯,弗雷
机械的酪氨酸的角色124年149年和丝氨酸UDP-galactose 4-epimerase从大肠杆菌。
生物化学。1997年9月2,36 (35):10675 - 84。
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9271498 (在PubMed]
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合成和超表达的基因编码大肠杆菌UDP-galactose 4-epimerase和改造,以促进盒式诱变。一般的酸碱催化活性位点的差向异构酶的动力学和光谱分析研究了野生型酶变异及其具体形式Y149F, S124A S124V, S124T。x射线晶体结构与UDP-glucose Y149F作为其流产的复杂的结构类似于相应的野生型复杂,除了149年酪氨酸酚氧的缺失。突变的主要影响表达的价值观kcat和kcat /公里。最活跃的突变Y149F, kcat的价值是野生型的0.010%的差向异构酶。S124A的活动也非常低,kcat值0.035%的原生酶。公里Y149F和S124A 12的值和21%的野生型酶,分别。S124T kcat约30%的价值的野生型酶,和价值的公里的原beplayapp生酶相似。UMP-dependent还原失活突变体的反应活性的葡萄糖也同样受到影响,与水被减少了6560 - 370年,和3.4倍Y149F S124A和S124T分别。NaBH3CN还原的二阶速率常数失活,而不需要一般碱催化,是类似的原生酶S124A, S124T, S124V。 However, Y149F reacts with NaBH3CN 12-20-fold faster than the wild-type enzyme at pH 8.5 and 7.0, respectively. The increased rate for Y149F is attributed to the weakened charge-transfer interaction between Phe 149 and NAD+, which is present with Tyr 149 in the wild-type enzyme. The charge-transfer band is present in the serine mutants, and its intensity at 320 nm is pH-dependent. The pH dependencies of A320 showed that the pKa values for Tyr 149 are 6.08 for the wild-type epimerase, 6.71 for S124A, 6.86 for S124V, and 6.28 for S124T. The low pKa value for Tyr 149 is attributed mainly to the positive electrostatic field created by NAD+ and Lys 153 (4.5 kcal mol-1) and partly to hydrogen bonding with Ser 124 (1 kcal mol-1). The pKa of Tyr 149 is the same as the kinetic pKa for the Bronsted base that facilitates hydride transfer to NAD+. We concluded that Tyr 149 provides the driving force for general acid-base catalysis, with Ser 124 playing an important role in mediating proton transfer.