Schwarz Alexandra, Pierfederici Francesco Maria, Nidetzky Bernd
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
Biochem J. 2005 Apr 15;387(Pt 2):437-45. doi: 10.1042/BJ20041593.
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 (His334-->Gln) and H334N mutants, and that of H334A was unchanged. In the direction of alpha-glucan synthesis, selectivity for the reaction with G1P (alpha-D-glucose 1-phosphate) compared with the selectivity for reaction with alpha-D-xylose 1-phosphate decreased from a wild-type value of approximately 20000 to 2600 and 100 in H334N and H334Q respectively. Binding of G1P 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 alpha-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; r2=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.
对一株月桂棒状杆菌淀粉磷酸化酶的定点突变体进行了纯化，其中His-334被丙氨酸、谷氨酰胺或天冬酰胺残基取代，通过对酶向磷酸转移糖基和从磷酸接受糖基的稳态动力学分析以及对配体与活性位点结合的研究对其进行了表征。与野生型相比，在30℃和pH 7.0条件下淀粉磷酸解的催化效率在H334Q（His334→Gln）和H334N突变体中分别下降了约150倍和50倍，而H334A的催化效率未变。在α-葡聚糖合成方向上，与α-D-木糖1-磷酸反应的选择性相比，与G1P（α-D-葡萄糖1-磷酸）反应的选择性从野生型的约20000分别降至H334N和H334Q中的2600和100。在突变体中，G1P与游离酶的结合减弱了10倍（H334N、H334Q）至50倍（H334A），而与酶和α-葡聚糖复合物的结合未受影响。在瞬态和稳态实验中，利用5'-磷酸吡哆醛辅因子荧光淬灭来检测抑制剂GL（D-葡萄糖酸1,5-内酯）与野生型和突变型酶的相互作用。GL与游离酶和酶-磷酸复合物的结合是一步完成的。与野生型相比，H334Q与磷酸结合的GL解离常数（K(d)）高出50倍是由于突变体中配体的解离速率增加所致。淀粉磷酸解的催化中心活性与倒数K(d)值的对数-对数相关性在一系列野生型和突变型酶中建立了线性自由能关系（斜率=1.19±0.07；r2=0.991）。这表明GL与磷酸结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起作用。
