Rowlett R, Yang L H, Ahmed S A, McPhie P, Jhee K H, Miles E W
Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Biochemistry. 1998 Mar 3;37(9):2961-8. doi: 10.1021/bi972286z.
Interaction between the alpha and beta subunits of tryptophan synthase leads to mutual stabilization of the active conformations and to coordinated control of the activities of the two subunits. To elucidate the roles of specific residues in the interaction site between the alpha and beta subunits, mutant alpha and beta subunits were constructed, and the effects of mutation on subunit interaction and intersubunit communication were determined. Mutation of either alpha subunit Asp56 (alphaD56A) or beta subunit Lys167 (betaK167T), residues that interact in some crystal structures of the tryptophan synthase alpha2beta2 complex, decreases the ability of the alpha subunit to activate the beta subunit and alters the reaction and substrate specificity of the beta subunit. Partial conformational repair is provided by alpha-glycerol 3-phosphate, a ligand that binds to the alpha subunit, or by Cs+ or NH4+, ligands that bind to the beta subunit. Mutation of beta subunit Arg175 (betaR175A), a residue that interacts with alpha subunit Pro57 in some structures, has much smaller effects on activity but results in a 15-fold increase in the apparent Kd for dissociation of the alpha and beta subunits. Replacement of the single tryptophan in the beta subunit by phenylalanine (W177F) has only small effects on activity but increases the apparent subunit dissociation constant approximately 10-fold. The most important conclusions of this investigation are that interaction between alphaAsp56 and betaLys167 is important for intersubunit communication and that mutual stabilization of the active conformations of the two subunits is impaired by mutation of either residue.
色氨酸合成酶的α亚基和β亚基之间的相互作用导致活性构象的相互稳定以及两个亚基活性的协同控制。为了阐明α亚基和β亚基之间相互作用位点中特定残基的作用，构建了突变的α亚基和β亚基，并确定了突变对亚基相互作用和亚基间通讯的影响。色氨酸合成酶α2β2复合物某些晶体结构中相互作用的残基，α亚基的天冬氨酸56（αD56A）或β亚基的赖氨酸167（βK167T）发生突变，会降低α亚基激活β亚基的能力，并改变β亚基的反应和底物特异性。α-甘油3-磷酸（一种与α亚基结合的配体）或Cs +或NH4 +（与β亚基结合的配体）可提供部分构象修复。β亚基的精氨酸175（βR175A）（在某些结构中与α亚基的脯氨酸57相互作用的残基）发生突变对活性的影响要小得多，但会导致α亚基和β亚基解离的表观解离常数（Kd）增加15倍。用苯丙氨酸（W177F）取代β亚基中的单个色氨酸对活性影响很小，但会使表观亚基解离常数增加约10倍。这项研究最重要的结论是，α天冬氨酸56和β赖氨酸167之间的相互作用对于亚基间通讯很重要，并且两个亚基活性构象的相互稳定会因任何一个残基的突变而受损。
