Abstract
Although alkaline phosphatase (APase) from Escherichia coli crystallizes as a symmetric dimer, it displays deviations from Michaelis–Menten kinetics supported by a model describing a dimeric enzyme with conformationally and kinetically non-equivalent subunits. The proposed model, explaining the mechanism of substrate hydrolysis, encompasses a conformational change mediated by subunit interactions [S. Orhanović, M. Pavela-Vrančič, Eur. J. Biochem. 270 (2003) 4356–4364]. The significance of interactions at the subunit interface and the involvement of the β-pleated sheet stretching from underneath the active site to the subunit surface, in the catalytic mechanism, has been probed by site-directed mutagenesis. The mutant APase, carrying alanine in place of Thr81, was analyzed in comparison to the wild-type protein. The T81A mutation, introduced at the subunit interface, significantly affected the protein kinetic properties, emphasizing the importance of subunit interactions in the catalytic process.
Published Version
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