A nucleophilic catalysis step is involved in the hydrolysis of aryl phosphate monoesters by human CT acylphosphatase.
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.
Paoli
Paolo
P
Department of Biochemical Sciences, University of Florence, Viale Morgagni 50, 50134 Florence, Italy.
Pazzagli
Luigia
L
Giannoni
Elisa
E
Caselli
Anna
A
Manao
Giampaolo
G
Camici
Guido
G
Ramponi
Giampietro
G
D017766:Acid Anhydride Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004952:Esters / Q000737:chemistry / Q000378:metabolism; D005990:Glycerol / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D009154:Mutation; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013329:Structure-Activity Relationship; D014867:Water / Q000737:chemistry; D000097024:Acylphosphatase
D017766:Acid Anhydride Hydrolases; D004952:Esters; D005990:Glycerol; D010710:Phosphates; D014867:Water; D000097024:Acylphosphatase