Limited proteolysis as a structural probe of the soluble alpha-glycerophosphate oxidase from Streptococcus sp.
Limited proteolysis as a structural probe of the soluble alpha-glycerophosphate oxidase from Streptococcus sp.
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有限的蛋白水解作为链球菌可溶性α-甘油磷酸氧化酶的结构探针。
DOI：
10.1021/bi992499j
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发表时间：
2000
期刊：
影响因子：
2.9
通讯作者：
Claiborne,A
中科院分区：
文献类型：
--
作者：
Charrier,V;Luba,J;Parsonage,D;Claiborne,A
如前所述[Parsonage，D.，Luba，J.，Mallett，T. C.的方法，和Claiborne，A.（1998）J.Biol.Chem.273，23812 - 23822]，来自许多肠球菌和链球菌来源的黄素蛋白α-甘油磷酸氧化酶（GlpO）含有在同源α-甘油磷酸氧化酶中完全不存在的保守的50 - 52残基插入物。在胰蛋白酶的限制性酶解作用下，从链球菌属（Streptococcussp.（m= 67.6 kDa）很容易转化为两个主要片段，对应于约40和23 kDa的质量。序列和质谱分析的联合应用表明，40-kDa片段代表完整GlpO的N-末端（Met 1 − Lys 368; 40.5 kDa），而23-kDa条带代表C-末端片段（Ala 405 − Lys 607; 22.9 kDa）。因此，有限的蛋白水解实际上切除了大部分GlpO插入片段（Ser 355-Lys 404），表明这代表了蛋白质表面上的柔性区域。酶的活性位点和其他光谱特性，包括黄素和色氨酸的荧光光谱，连二亚硫酸盐和亚硫酸盐的滴定行为，以及氧化黄素的阴离子形式的优先结合，在很大程度上不受蛋白水解的影响。对完整和有切口的链球菌GlpO（[GlpO] 10 μM）进行的酶监测周转分析表明，有切口的酶中的单一主要催化缺陷对应于Km（Glp）增加20倍;这种改变的动力学行为的基础来自于无氧停流实验中测量的切口酶还原的二级速率常数降低8倍。这些结果表明，由GlpO插入元件代表的柔性表面区域在介导有效的黄素还原中起着重要作用。
As reported previously [Parsonage, D., Luba, J., Mallett, T. C., and Claiborne, A. (1998)J. Biol. Chem.273, 23812−23822], the flavoprotein α-glycerophosphate oxidases (GlpOs) from a number of enterococcal and streptococcal sources contain a conserved 50−52 residue insert that is completely absent in the homologous α-glycerophosphate dehydrogenases. On limited proteolysis with trypsin, the GlpO fromStreptococcussp. (m= 67.6 kDa) is readily converted to two major fragments corresponding to masses of approximately 40 and 23 kDa. The combined application of sequence and mass spectrometric analyses demonstrates that the 40-kDa fragment represents the N-terminus of intact GlpO (Met1−Lys368; 40.5 kDa), while the 23-kDa band represents a C-terminal fragment (Ala405−Lys607; 22.9 kDa). Hence, limited proteolysis in effect excises most of the GlpO insert (Ser355−Lys404), indicating that this represents a flexible region on the protein surface. The active-site and other spectroscopic properties of the enzyme, including both flavin and tryptophan fluorescence spectra, titration behavior with both dithionite and sulfite, and preferential binding of the anionic form of the oxidized flavin, were largely unaffected by proteolysis. Enzyme-monitored turnover analyses of the intact and nicked streptococcal GlpOs (at [GlpO] ∼10 μM) demonstrate that the single major catalytic defect in the nicked enzyme corresponds to a 20-fold increase inKm(Glp); the basis for this altered kinetic behavior is derived from an 8-fold decrease in the second-order rate constant for reduction of the nicked enzyme, as measured in anaerobic stopped-flow experiments. These results indicate that the flexible surface region represented by elements of the GlpO insert plays an important role in mediating efficient flavin reduction.