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J Biol Chem, Vol. 273, Issue 16, 9602-9607, April 17, 1998

Re-evaluating the Role of His-143 in the Mechanism of Type I Dehydroquinase from Escherichia coli Using Two-dimensional ¹H,¹³C NMR

Andrew P. Leech, Ruth Boetzel^§, Colin McDonald^§, Annette K. Shrive^¶, Geoffrey R. Moore^§, John R. Coggins^**, Lindsay Sawyer^¶, and Colin Kleanthous

From the  School of Biological Sciences, ^§ School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom, ^¶ The Structural Biochemistry Group, Michael Swann Building, The University of Edinburgh, Edinburgh EH9 3JR, United Kingdom, and ^** Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, United Kingdom

Type I dehydroquinase from the shikimate pathway of Escherichia coli dehydrates dehydroquinate to dehydroshikimate. pH/log V_max profiles of the enzyme indicate the presence of a single ionizing group with a pK_a 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 pK_a 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 dehydroquinase. Following isotopic enrichment of wild-type and H143A dehydroquinase enzymes with [2-¹³C]histidine, the resonance for His-143 was assigned by comparing their ¹H,¹³C heteronuclear single quantum correlation NMR spectra. pH titrations revealed that whether in the liganded or unliganded state, His-143 does not ionize over the pH range 6-9.5 and so cannot possess a pK_a of 6.2. The NMR data are consistent with this residue remaining unprotonated at pH values optimal for the activity of this enzyme (pH > 7). The role of His-143 is re-evaluated in light of these and the recent structural data, and an alternative candidate for the pK_a of 6.2 is discussed.

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