Lolkema J S, ten Hoeve-Duurkens R H, Robillard G T
Eur J Biochem. 1986 Feb 3;154(3):651-6. doi: 10.1111/j.1432-1033.1986.tb09447.x.
Two interrelated sites have been detected on the fructose carrier in Rhodopseudomonas sphaeroides: an activity-linked dithiol and a Zn2+-binding site. Binding of Zn2+ brings EIIFru into a new conformation that to some extent mimics the conformation of phosphorylated EIIFru, an essential intermediate in the turnover of the enzyme. Binding of zinc to EIIFru or phosphorylating the enzyme protects it against trypsin inactivation relative to the dephosphorylated zinc-free enzyme. A dithiol is essential for activity. Interchanges between the redox states of the enzyme can be brought about by dithiothreitol and ferricyanide, but not, or very slowly, by molecular oxygen. The dithiol is protected, in the EIIFru-Zn2+ complex, against alkylation by MalNEt, reversible oxidation by Fe(CN)6(3-) and Cu2+, irreversible oxidation by Cu2+. The pK value of the activity linked thiol is 7.8. Protection experiments show that the dithiol is not located in any of the substrate-binding sites. The redox state of the enzyme does not influence the rate of inactivation of EIIFru by trypsin.
一个与活性相关的二硫醇和一个锌离子结合位点。锌离子的结合使EIIFru进入一种新的构象，这种构象在一定程度上模拟了磷酸化EIIFru的构象，而磷酸化EIIFru是该酶周转过程中的一个关键中间体。锌与EIIFru的结合或对该酶的磷酸化作用相对于去磷酸化的无锌酶而言，可保护其免受胰蛋白酶的失活作用。一种二硫醇对于活性至关重要。二硫苏糖醇和铁氰化物可导致该酶氧化还原状态之间的转换，但分子氧不能或非常缓慢地导致这种转换。在EIIFru-Zn2+复合物中，二硫醇受到保护，免受MalNEt的烷基化作用、Fe(CN)6(3-)和Cu2+的可逆氧化作用以及Cu2+的不可逆氧化作用。与活性相关的硫醇的pK值为7.8。保护实验表明，二硫醇并不位于任何底物结合位点。该酶的氧化还原状态并不影响胰蛋白酶对EIIFru的失活速率。
