可逆ligand-induced tryptophan-shift突变体的分离从杆菌stearothermophilus磷酸果糖激酶。
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朗宁博士,Riley-Lovingshimer先生Sacchettini JC,莱因哈特GD
可逆ligand-induced tryptophan-shift突变体的分离从杆菌stearothermophilus磷酸果糖激酶。
生物化学。2002年10月29日,41 (43):12967 - 74。
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12390023 (在PubMed]
- 文摘
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磷酸果糖激酶的tryptophan-shifted突变体的生物物理性质杆菌stearothermophilus (BsPFK)检查。突变,指定W179Y / Y164W,动力学和热力学性质类似于野生型酶。kcat下降了2倍观察,变异显示一个三倍小K(0.5)为基质,fructose-6-phosphate (Fru-6-P)比野生型酶。抑制剂的离解常数,磷烯醇丙酮酸(PEP),增加2倍,和耦合参数Q(喂),减少2倍。这表明,突变体显示略有降低PEP亲和力,一旦绑定PEP仍然是一个有效的抑制剂。色氨酸的新职位W179Y / Y164W大约是6 Fru-6-P部分的活性部位。观察到荧光强度下降了25%在Fru-6-P绑定,并观察到荧光强度减少80%与PEP绑定。此外,固有荧光偏振增加从0.327 + / - 0.001到0.353 + / - 0.001 Fru-6-P绑定,但是减少到0.290 + / - 0.001当PEP绑定。最值得注意的是,PEP的存在诱发四聚物的分离。四聚物的离解成二聚体发生在活性位点可以监控界面和活动或损失的损失色氨酸荧光时观察到酶与PEP滴定。 Activity can be protected or recovered by incubating the enzyme with Fru-6-P. Recovery of activity is enzyme concentration dependent, and the rate constant for association is 6.2 +/- 0.3 M(-1) x s(-1). Ultracentrifugation experiments revealed that in the absence of PEP the mutant enzyme exists in an equilibrium between the dimer and tetramer forms with a dissociation constant of 11.8 +/- 0.5 microM, while in the presence of PEP the enzyme exists in equilibrium between the dimer and monomer forms with a dissociation constant of 7.5 +/- 0.02 microM. A 3.1 A crystal structure of the mutant enzyme suggests that the amino acid substitutions have not dramatically altered the tertiary structure of the enzyme. While it is clear that wild-type BsPFK exists as a tetramer under these same conditions, these results suggest that quaternary structural changes probably play an important role in allosteric communication.