The anaerobic metabolism of phenol proceeds via carboxylation to 4-hydroxybenzoate by a two-step process involving seven proteins and two enzymes ("biological Kolbe-Schmitt carboxylation"). MgATP-dependent phosphorylation of phenol catalyzed by phenylphosphate synthase is followed by phenylphosphate carboxylation. Phenylphosphate synthase shows similarities to phosphoenolpyruvate (PEP) synthase and was studied for the bacterium Thauera aromatica. It consists of three proteins and transfers the beta-phosphoryl from ATP to phenol; the products are phenylphosphate, AMP, and phosphate. We showed that protein 1 becomes phosphorylated in the course of the reaction cycle by [beta-(32)P]ATP. This reaction requires protein 2 and is severalfold stimulated by protein 3. Stimulation of the reaction by 1 M sucrose is probably due to stabilization of the protein(s). Phosphorylated protein 1 transfers the phosphoryl group to phenolic substrates. The primary structure of protein 1 was analyzed by nanoelectrospray mass spectrometry after CNBr cleavage, trypsin digestion, and online high-pressure liquid chromatography at alkaline pH. His-569 was identified as the phosphorylated amino acid. We propose a catalytic ping-pong mechanism similar to that of PEP synthase. First, a diphosphoryl group is transferred to His-569 in protein 1, from which phosphate is cleaved to render the reaction unidirectional. Histidine phosphate subsequently serves as the actual phosphorylation agent.

译文

:苯酚的厌氧代谢通过包含7种蛋白质和2种酶的两步过程通过羧化反应生成4-羟基苯甲酸酯(“生物学Kolbe-Schmitt羧化反应”)。苯磷酸合酶催化苯酚的MgATP依赖性磷酸化,然后苯磷酸羧化。苯磷酸合酶显示与磷酸烯醇丙酮酸(PEP)合酶的相似性,并已针对芳香族拟南芥细菌进行了研究。它由三种蛋白质组成,将β-磷酰基从ATP转移到苯酚。产品是苯基磷酸酯,AMP和磷酸酯。我们显示蛋白质1在反应周期的过程中被[β-(32)P] ATP磷酸化。该反应需要蛋白质2,并被蛋白质3刺激几倍。1 M蔗糖刺激反应可能是由于蛋白质的稳定化所致。磷酸化的蛋白1将磷酸基团转移到酚类底物上。在碱性pH下,CNBr裂解,胰蛋白酶消化和在线高压液相色谱分析后,通过纳米电喷雾质谱分析蛋白质1的一级结构。 His-569被鉴定为磷酸化氨基酸。我们提出了一种类似于PEP合酶的催化乒乓机制。首先,二磷酰基被转移到蛋白质1中的His-569上,从中裂解磷酸盐使反应单向进行。磷酸组氨酸随后用作实际的磷酸化剂。

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