Escherichia coli glycerol kinase: role of a tetramer interface in regulation by fructose 1,6-bisphosphate and phosphotransferase system regulatory protein IIIglc.
Escherichia coli glycerol kinase: role of a tetramer interface in regulation by fructose 1,6-bisphosphate and phosphotransferase system regulatory protein IIIglc.
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大肠杆菌甘油激酶：四聚体界面在果糖 1,6-二磷酸和磷酸转移酶系统调节蛋白 IIIglc 调节中的作用。
DOI：
10.1021/bi00199a040
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Pettigrew,DW
中科院分区：
文献类型：
--
作者：
Liu,WZ;Faber,R;Feese,M;Remington,SJ;Pettigrew,DW
1994年6月13日修订的Mandarin pt接收·摘要：大肠杆菌甘油激酶（EC 2.7。1.30;可承诺量：甘油3-磷酸转移酶）是信号转导通路中的关键元件，该通路将甘油代谢所需的基因表达与甘油和葡萄糖的相对可用性偶联。它的催化活性受到蛋白质-蛋白质与Ills '0（一种磷酸转移酶系统蛋白）的相互作用和果糖1，6-二磷酸（FBP）的抑制;这些变构效应物中的每一种都构成葡萄糖可用的正信号。用羟胺诱变法筛选甘油激酶基因的突变体，以甘油代谢的葡萄糖抑制损失为指标。鉴定了两种突变酶，并通过DNA测序显示其含有丙氨酸65突变为苏氨酸（A65 T）和天冬氨酸72突变为天冬酰胺（D 72 N）。初速度的研究表明，突变并没有显着影响的催化性能，因此活性位点的结构，酶。两种突变均降低了FBP的抑制作用; A65 T消除了抑制作用，而D 72 N似乎降低了对FBP的亲和力和抑制程度。然而，这两个突变都不显著影响III 8 '0的抑制作用。凝胶渗透色谱研究表明，这两个突变改变二聚体-四聚体组装反应的酶和在增加分子量的FBP的效果。突变对组装反应的影响与这两个氨基酸残基在X射线结构中的位置一致，这表明它们与构成四聚体内两个亚基-亚基界面之一的螺旋相关。因此，影响甘油激酶二聚体-四聚体组装的突变降低了FBP的调节，但不改变III 8 '0的调节。这些结果表明，用于调节甘油激酶在体内的活性的变构机制可能依赖于其浓度。
Revised Manuscript Received June 13, 1994· abstract: Escherichia coli glycerol kinase (EC 2.7. 1.30; ATP: glycerol 3-phosphotransferase) is a key element in a signal transduction pathwaythat couples expression of genes required for glycerol metabolism to the relative availability of glycerol andglucose. Its catalytic activity is inhibited by protein-protein interactions with Ills'0, a phosphotransferase system protein, and by fructose 1, 6-bisphosphate (FBP); each of these allosteric effectors constitutes a positive signal that glucose is available. Loss of glucose inhibition of glycerol metabolism was used to screen for regulatory mutants of glycerol kinase after hydroxylamine mutagenesis of the cloned glpK gene. Two mutantenzymes were identified and shown by DNA sequencing to contain the mutations alanine 65 to threonine (A65T) and aspartate 72 to asparagine (D72N). Initial velocity studies show the mutations do not significantly affect the catalytic properties, hence active-site structures, of the enzymes. Both mutations decrease inhibition by FBP; A65T eliminates the inhibition while D72N appears to decrease the affinity for FBP and the extent of the inhibition. However, neither mutation significantly affects inhibition byIII8'0. Gel-permeation chromatography studies show that both of the mutations alter the dimer-tetramer assembly reaction of the enzyme and the effect of FBP in increasing the molecular weight. The effects of the mutations on the assembly reaction are consistent with the locations of these two amino acid residues in the X-ray structure, which shows them to be associated with an-helix that constitutes one of the two subunit-subunit interfaces within the tetramer. Thus, mutations that affect the dimer-tetramer assembly of glycerol kinase decrease the regulation by FBP but do not alter regulation byIII8'0. These results suggest that the allosteric mechanism used to regulate the activity of glycerol kinase in vivo may be dependent on its concentration.
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影响因子：
2.6
作者：
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通讯作者：
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影响因子：
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影响因子：
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作者：
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大肠杆菌甘油激酶的结晶和初步 X 射线研究。
影响因子：
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作者：
H. R. Faber;Donald W. Pettigrew;S. Remington
通讯作者：
S. Remington
影响因子：
2.9
作者：
BALDWIN, TO;BERENDS, T;ZIEGLER, MM
通讯作者：
ZIEGLER, MM