Evidence that mutations in a loop region of the alpha-subunit inhibit the transition from an open to a closed conformation in the tryptophan synthase bienzyme complex.
Evidence that mutations in a loop region of the alpha-subunit inhibit the transition from an open to a closed conformation in the tryptophan synthase bienzyme complex.
复制标题
有证据表明，α 亚基环区域的突变抑制色氨酸合酶双酶复合物从开放构象到闭合构象的转变。
DOI：
10.1016/s0021-9258(18)42377-0
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
M. Dunn
中科院分区：
文献类型：
--
作者：
P. Brzovic;Y. Sawa;C. Hyde;E. W. Miles;M. Dunn
采用快速扫描停流（RSSF）紫外可见光谱技术研究了鼠伤寒沙门氏菌色氨酸合成酶双酶复合物α -亚基单氨基酸突变对25 ~ 30 A远β -亚基活性位点反应性的影响。吡哆醛5'-磷酸（PLP）辅助因子提供了一种方便的光谱探针来直接监测β活性位点的催化事件。α亚基特异性配体α -甘油磷酸酯（GP）对plp依赖性β -反应的稳态和预稳态抑制作用被α亚基特异性配体α -甘油磷酸酯（GP）单次取代49号位置的Glu， 51号位置的Leu或60号位置的Asp强烈改变。然而，与野生型酶（Houben, k.f., and Dunn， M.F. (1990) Biochemistry 29, 2421-2429）所观察到的β位点共价中间体分布相似的gp诱导的变抗作用也在每种突变双酶复合物中被观察到。这些结果支持了一个假设，即吲哚从溶液进入β -位点的首选途径是通过α -位点和相互连接的通道（Dunn， M.F, Aguilar， V., brzoviki， P., Drewe， W.F, Houben， K.F, Leja， C.A和Roy， M.（1990）生物化学29,8598-8607）。残基α E49、α G51和α D60是α -亚基α / β桶拓扑结构中高度保守的插入序列的一部分。我们认为，gp诱导的β反应抑制部分是由于α -亚基的构象发生了依赖于配体的变化，从“开放”结构变为“封闭”结构，这涉及到α -亚基的这一区域，并阻碍了吲哚直接进入通道。我们的研究结果表明，在GP存在下观察到的α突变体的动力学行为改变反映了修饰的双酶复合物经历从开放到封闭形式的构象转变的能力受损。
Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy has been used to investigate the effects of single amino acid mutations in the alpha-subunit of the Salmonella typhimurium tryptophan synthase bienzyme complex on the reactivity at the beta-subunit active site located 25 to 30 A distant. The pyridoxal 5'-phosphate (PLP) cofactor provides a convenient spectroscopic probe to directly monitor catalytic events at the beta-active site. Single substitutions of Phe for Glu at position 49, Leu for Gly at position 51, or Tyr for Asp at position 60 in the alpha-subunit strongly alter the observed steady state and pre-steady state inhibitory effects of the alpha-subunit-specific ligand alpha-glycerophosphate (GP) on the PLP-dependent beta-reaction. However, similar GP-induced allosteric effects on the distribution of covalent intermediates bound at the beta-site that are observed with the wild-type enzyme (Houben, K.F., and Dunn, M.F. (1990) Biochemistry 29, 2421-2429) also are observed for each of the mutant bienzyme complexes. These results support the hypothesis that the preferred pathway of indole from solution into the beta-site is via the alpha-site and the interconnecting tunnel (Dunn, M.F., Aguilar, V., Brzović, P., Drewe, W.F., Houben, K.F., Leja, C.A., and Roy, M. (1990) Biochemistry 29, 8598-8607). Residues alpha E49, alpha G51, and alpha D60 are part of a highly conserved inserted sequence in the alpha/beta-barrel topology of the alpha-subunit. We propose that the GP-induced inhibition of the beta-reaction results, in part, from a ligand-dependent conformational change from an “open” to a “closed” structure of the alpha-subunit which involves this region of the alpha-subunit and serves to obstruct the direct access of indole into the tunnel. Our findings suggest that the altered kinetic behavior observed for the alpha-mutants in the presence of GP reflects an impaired ability of the modified bienzyme complex to undergo the conformational transition from the open to the closed form.