A Nucleophilic Catalysis Step is Involved in the Hydrolysis of
Aryl Phosphate Monoesters by Human CT Acylphosphatase*
Paolo
Paoli,
Luigia
Pazzagli,
Elisa
Giannoni,
Anna
Caselli,
Giampaolo
Manao,
Guido
Camici
, and
Giampietro
Ramponi
From the Department of Biochemical Sciences, University of
Florence, Viale Morgagni 50, 50134 Florence, Italy
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-H218O 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.
*
This work was funded in part by the Consiglio Nazionale
delle Ricerche (CNR Target Project on Biotechnology) and in part by the
Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR-CNR, Biotechnology Program L.95/95).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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