Identification of the essential histidine residue at the active site of Escherichia coli dehydroquinase.
The shikimate pathway enzyme 3-dehydroquinase is very susceptible to inactivation by the group-specific reagent diethyl pyrocarbonate (DEP). Inactivation follows pseudo first-order kinetics and exhibits a second-order rate constant of 148.5 M-1 min-1. An equilibrium mixture of substrate and product substantially protects against inactivation by DEP, suggesting that residues within the active site are being modified. Complete inactivation of the enzyme correlates with the modification of 6 histidine residues/subunit as determined by difference spectroscopy at 240 nm. Enzymic activity can be restored by hydroxylamine treatment, which is also consistent with the modification occurring at histidine residues. Using the kinetic method of Tsou (Tsou, C.-L. (1962) Sci. Sin. 11, 1535-1558), it was shown that modification of a single histidine residue leads to inactivation. Ligand protection experiments also indicated that 1 histidine residue was protected from DEP modification. pH studies show that the pKa for this inactivation is 6.18, which is identical to the single pKa determined from the pH/log Vmax profile for the enzyme. A single active site peptide was identified by differential peptide mapping in the presence and absence of ligand. This peptide was found to comprise residues 141-158; of the 2 histidines in this peptide (His-143 and His-146), only one, His-143, is conserved among all type I dehydroquinases. We propose that His-143 is the active site histidine responsible for DEP-mediated inactivation of dehydroquinase and is a good candidate for the general base that has been postulated to participate in the mechanism of this enzyme.
Deka
R K
RK
Department of Biochemistry, University of Glasgow, Scotland, United Kingdom.
Kleanthous
C
C
Coggins
J R
JR
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine / Q000737:chemistry; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D016415:Sequence Alignment
D008024:Ligands; D010446:Peptide Fragments; D006639:Histidine; D006836:Hydro-Lyases; C020000:3-dehydroquinate dehydratase; D004047:Diethyl Pyrocarbonate