Pettigrew D W, Smith G B, Thomas K P, Dodds D C
Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128, USA.
Arch Biochem Biophys. 1998 Jan 15;349(2):236-45. doi: 10.1006/abbi.1997.0444.
The structures of the sugar kinase/heat shock 70/actin superfamily of enzymes show that the active site is located in a deep cleft between two domains whose relative movement defines a domain closure conformational change thought to be involved in the catalytic and regulatory properties of members of the superfamily. To investigate the role of the domain closure in the regulatory behavior, site-directed mutagenesis is used to alter specific domain-domain interactions in Escherichia coli glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase), a member of this superfamily. Two active site aspartate residues are conserved throughout the superfamily, one (Asp245 in glycerol kinase) which is proposed to act as a general base during catalysis and one (Asp10 in glycerol kinase) which interacts with the Mg(II) ion of the bound Mg(II)-nucleotide complex. Each of these residues participates in domain-domain interactions that are mediated by the bound substrates. The enzymes containing the substitutions Asp245 to Asn (D245N) or Asp10 to Asn (D10N) were purified by affinity chromatography, and the effects of the substitutions on the catalytic properties and regulation by the allosteric effectors, fructose 1,6-bisphosphate (FBP), and the glucose-specific phosphocarrier protein, IIIGlc (also known as IIAGlc), were determined. Each of the residues participates in catalysis; kcat/Katp is decreased 300-fold by the D245N substitution and 100-fold by the D10N substitution. Affinity labeling with the glycerol analog 1,3-dichloroacetone shows that the level of activity seen for the D245N mutant enzyme is not due to deamidation of the substituted asparagine. Each of the substitutions has little effect on regulation by FBP and the apparent affinity for IIIGlc, and the D245N substitution does not affect the extent of inhibition by IIIGlc. However, the D10N substitution decreases the maximum extent of inhibition by IIIGlc from 100 to 60%, thus changing the action of IIIGlc to that of a partial inhibitor. The different sensitivities of the extents of FBP and IIIGlc inhibition to perturbation of a domain-domain interaction mediated by Asp10 suggest that the relations of the actions of these allosteric effectors to the domain closure conformational change are different.
糖激酶/热休克70/肌动蛋白超家族酶的结构表明，活性位点位于两个结构域之间的深裂隙中，这两个结构域的相对运动定义了一种结构域闭合构象变化，据认为这种变化与超家族成员的催化和调节特性有关。为了研究结构域闭合在调节行为中的作用，利用定点诱变来改变大肠杆菌甘油激酶（EC 2.7.1.30；ATP：甘油3-磷酸转移酶）（该超家族的一个成员）中特定的结构域-结构域相互作用。两个活性位点天冬氨酸残基在整个超家族中是保守的，一个（甘油激酶中的Asp245）在催化过程中被认为起通用碱的作用，另一个（甘油激酶中的Asp10）与结合的Mg（II）-核苷酸复合物的Mg（II）离子相互作用。这些残基中的每一个都参与由结合的底物介导的结构域-结构域相互作用。含有Asp245替换为Asn（D245N）或Asp10替换为Asn（D10N）的酶通过亲和层析纯化，并确定了这些替换对催化特性以及变构效应物果糖1,6-二磷酸（FBP）和葡萄糖特异性磷酸载体蛋白IIIGlc（也称为IIAGlc）调节的影响。每个残基都参与催化；D245N替换使kcat/Katp降低300倍，D10N替换使其降低100倍。用甘油类似物1,3-二氯丙酮进行亲和标记表明，D245N突变酶的活性水平不是由于被替换的天冬酰胺脱酰胺作用所致。每个替换对FBP调节和对IIIGlc的表观亲和力影响很小，并且D245N替换不影响IIIGlc的抑制程度。然而，D10N替换使IIIGlc的最大抑制程度从100%降至60%，从而将IIIGlc的作用改变为部分抑制剂的作用。FBP和IIIGlc抑制程度对由Asp10介导的结构域-结构域相互作用扰动的不同敏感性表明，这些变构效应物的作用与结构域闭合构象变化的关系是不同的。
