Catalytic mechanism of α-retaining glucosyl transfer by Corynebacterium callunae starch phosphorylase:: the role of histidine-334 examined through kinetic characterization of site-directed mutants
Catalytic mechanism of α-retaining glucosyl transfer by Corynebacterium callunae starch phosphorylase:: the role of histidine-334 examined through kinetic characterization of site-directed mutants
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DOI：
10.1042/bj20041593
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发表时间：
2005-04-15
影响因子：
4.1
通讯作者：
Nidetzky, B
中科院分区：
文献类型：
--
作者：
Schwarz, A;Pierfederici, FM;Nidetzky, B
纯化的callunae淀粉磷酸化酶，其中His-334被替换为丙氨酸，谷氨酰胺或天冬酰胺残基的定点突变体的特征在于通过稳态动力学分析的酶糖基转移到和从磷酸和研究的配体结合的活性位点。与野生型相比，在30 ℃和pH7.0条件下，淀粉的磷酸解催化效率降低了约10%。150-H334 Q（His（334）-> Gln）和H334 N突变体中的表达增加了50倍，而H334 A则没有变化。在α-葡聚糖合成的方向上，与α-D-木糖1-磷酸反应的选择性相比，与G1 P（α-D-葡萄糖1-磷酸）反应的选择性在H334 N和H334 Q中分别从类似于20000的野生型值降低至2600和100。在突变体中，GIP与游离酶的结合减弱了10倍（H334 N，H334 Q）和50倍（H334 A），而与酶和α-葡聚糖的复合物的结合不受影响。吡哆醛5 '-磷酸辅因子的荧光猝灭用于检测抑制剂GL（D-葡萄糖酸1，5-内酯）与野生型和突变型酶在瞬时和稳态实验中的相互作用。GL与游离酶和酶-磷酸盐复合物的结合在一个步骤中发生。与野生型相比，突变体中配体的解离速率增加，导致GL从与磷酸盐结合的H334 Q解离的常数（K-d）高50倍。淀粉磷酸解的催化中心活性与K-d值的倒数的对数-对数相关性建立了一个线性自由能关系（斜率= 1.19 +/- 0.07; r（2）= 0.991），在野生型和突变型酶的系列。它揭示了GL与磷酸盐的组合具有过渡态类似物的性质，并且His-334侧链在选择性稳定反应的过渡态中具有作用。
Purified site-directed mutants of Corynebacterium callunae starch phosphorylase in which His-334 was replaced by an alanine, glutamine or asparagine residue were characterized by steady-state kinetic analysis of enzymic glycosyl transfer to and from phosphate and studies of ligand binding to the active site. Compared with wild-type, the catalytic efficiencies for phosphorolysis of starch at 30 degrees C and pH 7.0 decreased approx. 150- and 50-fold in H334Q (His(334) -> Gln) and H334N mutants, and that of H334A was unchanged. In the direction of a-glucan synthesis, selectivity for the reaction with G1P (alpha-D-glucose 1-phosphate) compared with the selectivity for reaction with a-D-xylose 1-phosphate decreased from a wild-type value of similar to 20000 to 2600 and 100 in H334N and H334Q respectively. Binding of GIP to the free enzyme was weakened between 10-fold (H334N, H334Q) and 50-fold (H334A) in the mutants, whereas binding to the complex of enzyme and a-glucan was not affected. Quenching of fluorescence of the pyridoxal 5'-phosphate cofactor was used to examine interactions of the inhibitor GL (D-gluconic acid 1,5-lactone) with wild-type and mutant enzymes in transient and steady-state experiments. GL binding to the free enzyme and the enzyme-phosphate complex occurred in a single step. The 50-fold higher constant (K-d) for GL dissociation from H334Q bound to phosphate resulted from an increased off-rate for the ligand in the mutant, compared with wild-type. A log-log correlation of catalytic-centre activity for phosphorolysis of starch with a reciprocal K-d value established a linear free-energy relationship (slope = 1.19 +/- 0.07; r(2) = 0.991) across the series of wild-type and mutant enzymes. It reveals that GL in combination with phosphate has properties of a transition state analogue and that the His-334 side chain has a role in selectively stabilizing the transition state of the reaction.