Active site labeling of the shikimate pathway enzyme, dehydroquinase. Evidence for a common substrate binding site within dehydroquinase and dehydroquinate synthase.
Dehydroquinase, the third enzyme of the shikimate biosynthetic pathway, is inactivated by iodoacetate. Iodoacetate behaves as an affinity label for the Escherichia coli enzyme with a Ki of 30 mM and a limiting inactivation rate of 0.014 min-1 at pH 7.0 and 25 degrees C. Affinity labeling is mediated by the negative charge of the reagent since iodoacetamide does not inactivate the enzyme. 2.1-2.3 mol of carboxymethyl groups are incorporated per mol of protein monomer resulting in 90% inactivation of enzymic activity. The majority of the bound label (80%) is split equally between 2 methionine residues, Met-23 and Met-205, which were identified by sequencing radiolabelled peptide fragments isolated after proteolytic digestion. An equilibrium mixture of the substrate (dehydroquinate) and product (dehydroshikimate) substantially reduces the inactivation rate and specifically decreases the incorporation of label at both of these site, implicating them as being in or near the active site of the enzyme. Sequence alignments with other biosynthetic dehydroquinases show that of the 2 methionine residues only Met-205 is conserved. N-terminal alignments of all the available dehydroquinase sequences (both catabolic and biosynthetic classes) revealed that Met-23, although itself not conserved, resides within a cluster of conserved sequence which may constitute part of the dehydroquinate binding site. A consensus sequence was derived from these alignments and used to probe the protein sequence data banks. A related sequence was found in dehydroquinate synthase, the enzyme which precedes dehydroquinase in the shikimate pathway. These results suggest that we have identified part of the dehydroquinate binding site in both enzymes.
Kleanthous
C
C
Department of Biochemistry, University of Glasgow, Scotland.
Campbell
D G
DG
Coggins
J R
JR
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007461:Iodoacetates / Q000378:metabolism / Q000494:pharmacology*; D019807:Iodoacetic Acid; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000378:metabolism; D019761:Phosphorus-Oxygen Lyases; D012765:Shikimic Acid / Q000378:metabolism*
D000345:Affinity Labels; D007461:Iodoacetates; D010446:Peptide Fragments; D012765:Shikimic Acid; D008715:Methionine; D008190:Lyases; D006836:Hydro-Lyases; C020000:3-dehydroquinate dehydratase; C020001:3-dehydroquinate synthetase; D019761:Phosphorus-Oxygen Lyases; D019807:Iodoacetic Acid