The axial ligand of the catalytic mononuclear T1 copper site (Met502) of the CotA laccase was replaced by a leucine or phenylalanine residue to increase the redox potential of the enzyme. These mutations led to an increase in the redox potential by approx. 100 mV relative to the wild-type enzyme but the catalytic constant kcat in the mutant enzymes was severely compromised. This decrease in the catalytic efficiency was unexpected as the X-ray analysis of mutants has shown that replacement of methionine ligand did not lead to major structural changes in the geometry of the T1 centre or in the overall fold of the enzyme. However, the mutations have a profound impact on the thermodynamic stability of the enzyme. The fold of the enzyme has become unstable especially with the introduction of the larger phenylalanine residue and this instability should be related to the decrease in the catalytic efficiency. The instability of the fold for the mutant proteins resulted in the accumulation of an intermediate state, partly unfolded, in-between native and unfolded states. Quenching of tryptophan fluorescence by acrylamide has further revealed that the intermediate state is partly unfolded.
Conference Article| November 23 2007
Insight into stability of CotA laccase from the spore coat of Bacillus subtilis
E.P. Melo 1
*Instituto de Biotecnologia e Bioengenharia, Centro de Biomedicina Molecular e Estrutural, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal
†Instituto de Biotecnologia e Bioengenharia, Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
1To whom correspondence should be addressed, at Instituto de Biotecnologia e Bioengenharia, Centro de Biomedicina Molecular e Estrutural, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal (email email@example.com).
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E.P. Melo, A.T. Fernandes, P. Durão, L.O. Martins; Insight into stability of CotA laccase from the spore coat of Bacillus subtilis. Biochem Soc Trans 1 December 2007; 35 (6): 1579–1582. doi: https://doi.org/10.1042/BST0351579
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