J. Biochem, 2004, Vol. 135, No. 6 721-730
© 2004 The Japanese Biochemical Society
BIOCHEMISTRY |
Quantitative Structure-Activity Relationship for the Cleavage of C3/C4-Substituted Catechols by a Prototypal Extradiol Catechol Dioxygenase with Broad Substrate Specificity
Department of Biochemistry, Shiga University of Medical Science, Seta, Ohtsu, Shiga 520-2192
Catechol 2,3-dioxygenase [EC 1.13.11.2] from Pseudomonas putida mt-2 (Mpc) catalyzes the extradiol cleavage of catechol to produce 2-hydroxymuconate semialdehyde. The Km values for the catecholic substrate (KmA) and O2 (KmO2), and catalytic constants (kcat) were kinetically determined for eight C3/C4-substituted catechols at 25°C and pH 6.5 or 7.5. The first pKa values (pK1) were determined for eleven catechols (pK1 = 7.26–9.47), correlated with Hammett substituent constants, and electron-withdrawing substituents significantly stabilized the monoanionic species of free catechols. Mpc preferred catechols with non-ionic substituents at the C3 or C4 position. 3-Phenylcatechol, a biphenyl, was cleaved, while 4-tert-butylcatechol was not. The logarithm of kcat/KmA (substrate specificity constant) exhibited a good linear correlation with pK1, with the exception of those for 4-halocatechols. The logarithm of kcat/KmO2 showed a good linear correlation with pK1, with the exception of that of 3-phenylcatechol. These results demonstrate that catechol binding to the Mpc active site, the following O2 binding, and the activation of the bound O2 are all sensitive to electronic effects of the substituents. However, kcat did not correlate significantly with pK1. The present study distinguishes clearly between the electronic and the steric effects of catecholic substrates in the reactivity of Mpc, and provides important insight into the mechanistic basis for a vast range of substrate specificities of extradiol dioxygenases.
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