Re-evaluating the role of His-143 in the mechanism of type I dehydroquinase from Escherichia coli using two-dimensional 1H,13C NMR.

The Journal of Biological Chemistry
A P LeechC Kleanthous

Abstract

Type I dehydroquinase from the shikimate pathway of Escherichia coli dehydrates dehydroquinate to dehydroshikimate. pH/log Vmax profiles of the enzyme indicate the presence of a single ionizing group with a pKa of 6.2. Chemical modification experiments with diethyl pyrocarbonate have identified the conserved residue His-143 as essential for catalysis in this enzyme and the pKa for this modification is also 6.2, implying that this is the single ionizing residue in dehydroquinase that may be acting as a general base in the catalytic mechanism. Subsequent mutagenesis of this residue (Leech, A. P., James, R., Coggins, J. R., and Kleanthous, C. (1995) J. Biol. Chem. 270, 25827-25836) further suggested that His-143 may be involved in Schiff base formation/breakdown as well as being the proton abstracting general base. The importance of this residue was confirmed by recent x-ray crystallographic data showing His-143 to be at the center of a hydrogen-bonded triad, flanked by the essential Schiff base forming residue Lys-170 and Glu-86. In the present study, we have used mutagenesis and 1H and 13C NMR to assign the resonance of His-143 and probe its ionization state to define more precisely its role in the mechanism of type I dehydroqui...Continue Reading

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