Effects of surface hydrophobicity on the catalytic iron ion retention in the active site of two catechol 1,2-dioxygenase isoenzymes.
The different behaviour of two isozymes (IsoA and IsoB) of catechol 1,2-dioxygenase (C 1,20) from Acinetobacter radioresistens S13 on a hydrophobic interaction, Phenyl-Sepharose chromatographic column, prompted us to investigate the role of superficial hydrophobicity on structural-functional aspects for such class of enzymes. The interaction of 8-anilino-1-naphtalenesulphonate (ANS), a fluorescent probe known to bind to hydrophobic sites in proteins, revealed that the two isoenzymes have a markedly different hydrophobicity degree although a similar number of hydrophobic superficial sites were estimated (2.65 for IsoA and 2.18 for IsoB). ANS is easily displaced by adding the substrates catechol or 3-methylcatechol to the adduct, suggesting that the binding sites are in the near surroundings of the catalytic clefts. The analysis of the hydropathy profiles and the possible superficial cavities allowed to recognize the most feasible region for ANS binding. The lower hydrophobicity detected in the near surroundings of the catalytic pocket of IsoB supports its peculiarity to lose the catalytic metal ions more easily than IsoA. As previously suggested for other metalloenzymes, the presence of more hydrophilic and/or smaller residues near to the active site of IsoB is expected to increase the metal ligands mobility thus increasing the metal ion dissociation rate constants, estimated to be 0.078 h(-1) and 0.670 h(-1) for IsoA and IsoB respectively.
Di Nardo
Giovanna
G
Dipartimento di Biologia Animale e dell'Uomo, Università di Torino, Via Accademia Albertina 13, 10123 Torino, Italy. giovanna.dinardo@unito.it
Pessione
Enrica
E
Cavaletto
Maria
M
Anfossi
Laura
L
Vanni
Adriano
A
Briganti
Fabrizio
F
Giunta
Carlo
C
D039903:Actinobacteria; D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D001665:Binding Sites; D002138:Calibration; D002384:Catalysis; D020134:Catalytic Domain; D002396:Catechols / Q000737:chemistry; D049308:Dioxygenases / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D006358:Hot Temperature; D007477:Ions; D007501:Iron / Q000737:chemistry*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000737:chemistry; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013054:Spectrophotometry, Atomic; D013696:Temperature; D013997:Time Factors
D000817:Anilino Naphthalenesulfonates; D002396:Catechols; D007477:Ions; C027132:1-anilino-8-naphthalenesulfonate; D007501:Iron; D006899:Mixed Function Oxygenases; D010105:Oxygenases; D049308:Dioxygenases; C022336:phenol 2-monooxygenase; C034221:catechol