Side reactions catalyzed by ribulose-bisphosphate carboxylase in the presence and absence of small subunits
Side reactions catalyzed by ribulose-bisphosphate carboxylase in the presence and absence of small subunits
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DOI：
10.1074/jbc.272.9.5445
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发表时间：
1997-02-28
影响因子：
4.8
通讯作者：
Andrews, TJ
中科院分区：
文献类型：
--
作者：
Morell, MK;Wilkin, JM;Andrews, TJ
在大肠杆菌中表达的来自聚球藻属PCC 6301的核酮糖-二磷酸羧化酶的大亚基核心在其小亚基不存在的情况下保留了痕量的羧化酶活性（全酶的k（cat）的约1%）（Andrews，T. J（1988）J.Biol.Chem.263，12213-12219），在底物饱和的稳态催化过程中，由于无机磷酸盐从第一反应中间体（核酮糖二磷酸的2，3-烯醇形式）中的β消除，该残余活性将约10%的反应通量转移到1-脱氧-D-甘油基-2，3-戊二酮糖-5-磷酸。这表明活性位点稳定和/或保留该中间体的能力由于缺乏小亚基而受到损害。由于缺乏小亚基，由烯醇化中间体的错误质子化引起的底物的差向异构化和异构化没有显著加剧。残留的羧化活性以类似于全酶的比率在丙酮酸和3-磷酸甘油酸之间分配产物，这表明倒数第二个三碳酸-酸中间体的稳定化不受缺乏小亚基的干扰。揭示了潜在的不稳定性的五碳englycol中间体，即使与全酶，在设计的条件下，导致耗尽底物CO2（和O-2），当羧化（和氧合）停止后，耗尽气态底物，菠菜和聚球藻全酶继续缓慢β消除无机磷酸盐和misprotonate englycol中间体。随着羧化和氧化作用的阻断，这些包被中间体的副反应的产物积累到容易检测的水平，说明了核酮糖-P-2羧化酶使用这种反应性物质作为催化中间体的困难。
The large subunit core of ribulose-bisphosphate carboxylase from Synechococcus PCC 6301 expressed in Escherichia coli in the absence of its small subunits retains a trace of carboxylase activity (about 1% of the k(cat) of the holoenzyme) (Andrews, T. J (1988) J. Biol. Chem. 263, 12213-12219), During steady-state catalysis at substrate saturation, this residual activity diverted approximately 10% of the reaction flux to 1-deoxy-D-glycero-2,3-pentodiulose-5-phosphate as a result of beta elimination of inorganic phosphate from the first reaction intermediate, the 2,3-enediol form of ribulose bisphosphate. This indicates that the active site's ability to stabilize and/or retain this intermediate is compromised by the absence of small subunits, Epimerization and isomerization of the substrate resulting from misprotonation of the enediol intermediate were not significantly exacerbated by lack of small subunits, The residual carboxylating activity partitioned product between pyruvate and 3-phosphoglycerate in a ratio similar to that of the holoenzyme, indicating that stabilization of the penultimate three-carbon aci-acid intermediate is not perturbed by lack of small subunits. The underlying instability of the five-carbon enediol intermediate was revealed, even with the holoenzyme, under conditions designed to lead to exhaustion of substrate CO2 (and O-2), When carboxylation (and oxygenation) stalled upon exhaustion of gaseous substrate, both spinach and Synechococcus holoenzymes continued slowly to beta eliminate inorganic phosphate from and to misprotonate the enediol intermediate. With carboxylation and oxygenation blocked, the products of these side reactions of the enediol intermediate accumulated to readily detectable levels, illustrating the difficulties attendant upon ribulose-P-2 carboxylase's use of this reactive species as a catalytic intermediate.