Paoli Paolo, Pazzagli Luigia, Giannoni Elisa, Caselli Anna, Manao Giampaolo, Camici Guido, Ramponi Giampietro
Department of Biochemical Sciences, University of Florence, Viale Morgagni 50, 50134 Florence, Italy.
J Biol Chem. 2003 Jan 3;278(1):194-9. doi: 10.1074/jbc.M206918200. Epub 2002 Oct 29.
Acylphosphatase, one of the smallest enzymes, is expressed in all organisms. It displays hydrolytic activity on acyl phosphates, nucleoside di- and triphosphates, aryl phosphate monoesters, and polynucleotides, with acyl phosphates being the most specific substrates in vitro. The mechanism of catalysis for human acylphosphatase (the organ-common type isoenzyme) was investigated using both aryl phosphate monoesters and acyl phosphates as substrates. The enzyme is able to catalyze phosphotransfer from p-nitrophenyl phosphate to glycerol (but not from benzoyl phosphate to glycerol), as well as the inorganic phosphate-H(2)18O oxygen exchange reaction in the absence of carboxylic acids or phenols. In short, our findings point to two different catalytic pathways for aryl phosphate monoesters and acyl phosphates. In particular, in the aryl phosphate monoester hydrolysis pathway, an enzyme-phosphate covalent intermediate is formed, whereas the hydrolysis of acyl phosphates seems a more simple process in which the Michaelis complex is attacked directly by a water molecule generating the reaction products. The formation of an enzyme-phosphate covalent complex is consistent with the experiments of isotope exchange and transphosphorylation from substrates to glycerol, as well as with the measurements of the Brønsted free energy relationships using a panel of aryl phosphates with different structures. His-25 involvement in the formation of the enzyme-phosphate covalent complex during the hydrolysis of aryl phosphate monoesters finds significant confirmation in experiments performed with the H25Q mutated enzyme.
酰基磷酸酶是最小的酶之一，在所有生物体中都有表达。它对酰基磷酸酯、核苷二磷酸和三磷酸、芳基磷酸单酯以及多核苷酸具有水解活性，其中酰基磷酸酯是体外最具特异性的底物。使用芳基磷酸单酯和酰基磷酸酯作为底物，对人酰基磷酸酶（器官通用型同工酶）的催化机制进行了研究。该酶能够催化磷酸从对硝基苯磷酸酯转移至甘油（但不能从苯甲酰磷酸酯转移至甘油），以及在不存在羧酸或酚类的情况下进行无机磷酸-H₂¹⁸O氧交换反应。简而言之，我们的研究结果表明芳基磷酸单酯和酰基磷酸酯存在两种不同的催化途径。特别是，在芳基磷酸单酯水解途径中，会形成酶-磷酸共价中间体，而酰基磷酸酯的水解似乎是一个更简单的过程，其中米氏复合物直接被水分子攻击生成反应产物。酶-磷酸共价复合物的形成与同位素交换和从底物到甘油的转磷酸化实验一致，也与使用一组具有不同结构的芳基磷酸酯测量的布仑斯惕自由能关系一致。在使用H25Q突变酶进行的实验中，His-25参与芳基磷酸单酯水解过程中酶-磷酸共价复合物的形成得到了有力证实。
