Rat kidney acylase I: further characterisation and mutation studies on the involvement of Glu 147 in the catalytic process.
Rat kidney acylase I was characterised by performing site-directed mutagenesis and enzymatic analysis in the presence of various chemical inhibitors. Site-directed mutagenesis on E147 and overexpression of the protein in a bacterial system, revealed the importance of this residue in enzymatic activity, it corresponds to the putative catalytic E175 in carboxypeptidase G2 from Pseudomonas aeruginosa. The reactivity of histidine and cysteine residues of acylase I with diethylpyrocarbonate (DEPC) and mercuric chloride, respectively, showed that these two amino acids are required for the enzyme to be fully active. Interestingly, the effects of mercuric chloride on rat kidney acylase I were not as great as those on the porcine enzyme, in agreement with previously observed differences between the two enzymes. Moreover, N-[3-(2-furyl)-acryloyl-L-methionine] (FA-Met) a synthetic substrate of the porcine acylase I was found to be an inhibitor of the rat kidney enzyme. These results strongly suggest the existence of differences between the active site of rat and porcine kidney acylases I. Lastly, the rat kidney enzyme was as sensitive as its porcine counterpart to two metal chelating agents, 1,10-phenanthroline and ethylenediamine tetraacetate (EDTA).
Durand
Anne
A
Faculté des Sciences et Techniques Saint-Jérôme, Institut Méditerranéen de Recherche en Nutrition, Service 342, Umr Université Aix-Marseille III-INRA 1111, avenue Escadrille-Normandie-Niemen, 13397 Marseille cedex 20, France.
Giardina
Thierry
T
Villard
Claude
C
Roussel
Alain
A
Puigserver
Antoine
A
Perrier
Josette
J
D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D008627:Mercuric Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010618:Phenanthrolines / Q000494:pharmacology; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013552:Swine
D010618:Phenanthrolines; D011994:Recombinant Proteins; D018698:Glutamic Acid; D008627:Mercuric Chloride; D004492:Edetic Acid; D000581:Amidohydrolases; C019672:aminoacylase I; D004047:Diethyl Pyrocarbonate; C025205:1,10-phenanthroline