Gottlin E B, Rudolph A E, Zhao Y, Matthews H R, Dixon J E
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Proc Natl Acad Sci U S A. 1998 Aug 4;95(16):9202-7. doi: 10.1073/pnas.95.16.9202.
The phospholipase D (PLD) superfamily includes enzymes of phospholipid metabolism, nucleases, as well as ORFs of unknown function in viruses and pathogenic bacteria. These enzymes are characterized by the invariant sequence motif, H(X)K(X)4D. The endonuclease member Nuc of the PLD family was over-expressed in bacteria and purified to homogeneity. Mutation of the conserved histidine to an asparagine in the endonuclease reduced the kcat for hydrolysis by a factor of 10(5), suggesting that the histidine residue plays a key role in catalysis. In addition to catalyzing hydrolysis, a number of phosphohydrolases will catalyze a phosphate (oxygen)-water exchange reaction. We have taken advantage of this observation and demonstrate that a 32P-labeled protein could be trapped when the enzyme was incubated with 32P-labeled inorganic phosphate. The phosphoenzyme intermediate was stable in 1 M NaOH and labile in 1 M HCl and 1 M hydroxylamine, suggesting that the enzyme forms a phosphohistidine intermediate. The pH-stability profile of the phosphoenzyme intermediate was consistent with phosphohistidine and the only radioactive amino acid found after alkaline hydrolysis was phosphohistidine. These results suggest that the enzymes in the PLD superfamily use the conserved histidine for nucleophilic attack on the substrate phosphorus atom and most likely proceed via a common two-step catalytic mechanism.
磷脂酶D（PLD）超家族包括磷脂代谢酶、核酸酶，以及病毒和致病细菌中功能未知的开放阅读框。这些酶的特征在于保守序列基序H(X)K(X)4D。PLD家族的核酸酶成员Nuc在细菌中过表达并纯化至同质。核酸酶中保守的组氨酸突变为天冬酰胺使水解的催化常数降低了10^5倍，表明组氨酸残基在催化中起关键作用。除了催化水解外，许多磷酸水解酶还将催化磷酸（氧）-水交换反应。我们利用了这一观察结果，并证明当酶与32P标记的无机磷酸盐一起孵育时，32P标记的蛋白质可以被捕获。磷酸化酶中间体在1 M NaOH中稳定，在1 M HCl和1 M羟胺中不稳定，表明该酶形成磷酸组氨酸中间体。磷酸化酶中间体的pH稳定性曲线与磷酸组氨酸一致，碱性水解后发现的唯一放射性氨基酸是磷酸组氨酸。这些结果表明，PLD超家族中的酶利用保守的组氨酸对底物磷原子进行亲核攻击，并且很可能通过常见的两步催化机制进行。
