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首页> 外文OA文献 >Substrate binding and catalysis in carbamate kinase ascertained by crystallographic and site-directed mutagenesis studies: movements and significance of a unique globular subdomain of this key enzyme for fermentative ATP production in bacteria
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Substrate binding and catalysis in carbamate kinase ascertained by crystallographic and site-directed mutagenesis studies: movements and significance of a unique globular subdomain of this key enzyme for fermentative ATP production in bacteria

机译：通过结晶学和定点诱变研究确定的氨基甲酸酯激酶的底物结合和催化作用：该关键酶的独特球状亚结构域的运动及其在细菌中发酵产生ATP的意义

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摘要

Carbamate kinase (CK) makes ATP from ADP and carbamoyl phosphate (CP) in the final step of the microbial fermentative catabolism of arginine, agmatine, and oxalurate/allantoin. Two previously reported CK structures failed to clarify CP binding and catalysis and to reveal the significance of the protruding subdomain (PSD) that hangs over the CK active center as an exclusive and characteristic CK feature. We clarify now these three questions by determining two crystal structures of Enterococcus faecalis CK (one at 1.5 A resolution and containing bound MgADP, and the other at 2.1 A resolution and having in the active center one sulfate and two fixed water molecules that mimic one bound CP molecule) and by mutating active-center residues, determining the consequences of these mutations on enzyme functionality. Superimposition of the present crystal structures reconstructs the filled active center in the ternary complex, immediately suggesting in-line associative phosphoryl group transfer and a mechanism for enzyme catalysis involving N51, K209, K271, D210, and the PSD residue K128. The large respective increases and decreases in K(m)(CP) and k(cat) triggered by the mutations N51A, K128A, K209A, and D210N corroborate the ternary complex active-site architecture and the catalytic mechanism proposed. The extreme negative effects of K128A demonstrate a key role of the PSD in substrate binding and catalysis. The crystal structures reveal large rigid-body movements of the PSD towards the enzyme body that place K128 next to CP and bury the CP site. A mechanism that connects CP site occupation with the PSD approach, involving V206-I207 in the CP site and P162-S163 in the PSD stem, is identified. The effects of the V206A and V206L mutations support this mechanism. It is concluded that the PSD movement allows CK to select against the abundant CP/carbamate analogues acetylphosphate/acetate and bicarbonate, rendering CK highly selective for CP/carbamate.
机译：氨基甲酸酯激酶（CK）在精氨酸，胍丁胺和草酸/丙二酸的微生物发酵分解代谢的最后步骤中，由ADP和氨基甲酰磷酸（CP）生成ATP。先前报道的两个CK结构未能阐明CP的结合和催化作用，也没有揭示悬垂在CK活性中心上的突出子结构域（PSD）作为CK独有和特征性特征的重要性。现在，我们通过确定粪肠球菌CK的两个晶体结构（一个以1.5 A的分辨率并包含结合的MgADP，另一个以2.1 A的分辨率并在活性中心具有一个硫酸盐和两个模拟一个键的固定水分子，来阐明这三个问题。 CP分子）并通过突变活性中心残基来确定这些突变对酶功能的影响。本晶体结构的叠加重建了三元复合物中填充的活性中心，立即表明了在线缔合磷酸基团的转移以及涉及N51，K209，K271，D210和PSD残基K128的酶催化机制。由突变N51A，K128A，K209A和D210N引发的K（m）（CP）和k（cat）的较大的分别增加和减少，证实了三元复合物活性位点结构和所提出的催化机理。 K128A的极端负面影响证明了PSD在底物结合和催化中的关键作用。晶体结构揭示了PSD向酶体的大刚体运动，使K128靠近CP并掩埋了CP位点。确定了一种将CP站点占用与PSD方法联系起来的机制，其中涉及CP站点中的V206-I207和PSD茎中的P162-S163。 V206A和V206L突变的影响支持这种机制。结论是PSD运动允许CK选择对抗大量CP /氨基甲酸酯类似物乙酰磷酸盐/乙酸盐和碳酸氢盐，从而使CK对CP /氨基甲酸酯具有高度选择性。

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