Authors: Ishida T.; Tanaka H.; Horiike K.

Source: The Journal of Biochemistry, Volume 135, Number 6, June 2004 , pp. 721-730(10)

Publisher: Oxford University Press

Abstract:

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 K<inf>m</inf> values for the catecholic substrate (K<inf>mA</inf>) and O<inf>2</inf> (K<inf>mO2</inf>), and catalytic constants (k<inf>cat</inf>) were kinetically determined for eight C3/C4-substituted catechols at 25°C and pH 6.5 or 7.5. The first pK<inf>a</inf> values (pK<inf>1</inf>) were determined for eleven catechols (pK<inf>1</inf> = 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 k<inf>cat</inf>/K<inf>mA</inf> (substrate specificity constant) exhibited a good linear correlation with pK<inf>1</inf>, with the exception of those for 4-halocatechols. The logarithm of k<inf>cat</inf>/K<inf>mO2</inf> showed a good linear correlation with pK<inf>1</inf>, with the exception of that of 3-phenylcatechol. These results demonstrate that catechol binding to the Mpc active site, the following O<inf>2</inf> binding, and the activation of the bound O<inf>2</inf> are all sensitive to electronic effects of the substituents. However, k<inf>cat</inf> did not correlate significantly with pK<inf>1</inf>. 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.

Document Type: Research article

DOI: http://dx.doi.org/10.1093/jb/mvh089

Publication date: 2004-06-01

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