Archael phosphoproteins. Identification of a hexosephosphate mutase and the alpha-subunit of succinyl-CoA synthetase in the extreme acidothermophile Sulfolobus solfataricus.
Archael phosphoproteins. Identification of a hexosephosphate mutase and the alpha-subunit of succinyl-CoA synthetase in the extreme acidothermophile Sulfolobus solfataricus.
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古细菌磷蛋白。
DOI：
10.1002/pro.5560070111
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Kennelly,PJ
中科院分区：
文献类型：
--
作者：
Solow,B;Bischoff,KM;Zylka,MJ;Kennelly,PJ
当极端嗜酸性古菌硫磺硫化叶菌的可溶性提取物与[γ-32 P]ATP孵育时，在SDS-PAGE后观察到几种放射性标记的多肽。其中最突出的迁移表观分子量为14，18，35，42，46，50和79 kDa。磷酸氨基酸分析显示，所有蛋白质都含有磷酸丝氨酸，除了35-kDa的蛋白质，其蛋白质-磷酸连接被证明对强酸不稳定。所观察到的磷酸化模式的影响所使用的二价金属离子辅因子的身份，Mg 2+与Mn 2+，和孵育温度的选择。纯化35-和50-kDa磷蛋白，并测定其氨基末端序列。前一种多肽的氨基末端序列与琥珀酰辅酶A合成酶α亚基的保守部分紧密匹配，后者在其催化循环期间形成酸不稳定的磷酸组氨酰酶中间体。琥珀酸盐或ADP特异性去除放射性标记的能力证实了这一鉴别。50-kDa多肽的序列含有七肽基序，Phe/Pro-Gly-Thr-Asp/Ser-Gly-瓦尔/Leu-Arg，在几种磷酸己糖变位酶中的相似位置发现。这些变位酶的催化机制涉及形成磷酸丝氨酰酶中间体。通过（1）糖和糖磷酸盐诱导从蛋白质中去除标记的磷酰基的能力，以及（2）[32 P]葡萄糖6-磷酸盐将其磷酰基捐赠给蛋白质的能力，证实了p50作为磷酸己糖的身份。
When soluble extracts from the extreme acidophilic archaeonSulfolobus solfataricuswere incubated with [γ‐32P]ATP, several radiolabeled polypeptides were observed following SDS‐PAGE. The most prominent of these migrated with apparent molecular masses of 14, 18, 35, 42, 46, 50, and 79 kDa. Phosphoamino acid analysis revealed that all of the proteins contained phosphoserine, with the exception of the 35‐kDa one, whose protein‐phosphate linkage proved labile to strong acid. The observed pattern of phosphorylation was influenced by the identity of the divalent metal ion cofactor used, Mg2+versus Mn2+, and the choice of incubation temperature. The 35‐ and 50‐kDa phosphoproteins were purified and their amino‐terminal sequences determined. The former polypeptide's amino‐terminal sequence closely matched a conserved portion of the α‐subunit of succinyl‐CoA synthetase, which forms an acid‐labile phosphohistidyl enzyme intermediate during its catalytic cycle. This identification was confirmed by the ability of succinate or ADP to specifically remove the radiolabel. The 50‐kDa polypeptide's sequence contained a heptapeptide motif, Phe/Pro‐Gly‐Thr‐Asp/Ser‐Gly‐Val/Leu‐Arg, found in a similar position in several hexosephosphate mutases. The catalytic mechanism of these mutases involves formation of a phosphoseryl enzyme intermediate. The identity of p50 as a hexosephosphate mutase was confirmed by (1) the ability of sugars and sugar phosphates to induce removal of the labeled phosphoryl group from the protein, and (2) the ability of [32P]glucose 6‐phosphate to donate its phosphoryl group to the protein.