Localization of the Active Site of Type II Dehydroquinases

Abstract

A novel method based on electrospray mass spectrometry (Krell, T., Pitt, A. R., and Coggins, J. R. (1995) FEBS Lett. 360, 93-96) has been used to localize active site residues in the type I and type II dehydroquinases. Both enzymes have essential hyper-reactive arginine residues, and the type II enzymes have an essential tyrosine residue. The essential hyper-reactive Arg-23 of the Streptomyces coelicolor type II enzyme has been replaced by lysine, glutamine, and alanine residues. The mutant enzymes were purified and shown by CD spectroscopy to be structurally similar to the wild-type enzyme. All three mutant enzymes were much less active, for example the k cat of the R23A mutant was 30,000-fold reduced. The mutants all had reduced K m values, indicating stronger substrate binding, which was confirmed by isothermal titration calorimetry experiments. A role for Arg-23 in the stabilization of a carbanion intermediate is proposed. Comparison of the amino acid sequence around the hyper-reactive arginine residues of the two classes of enzymes indicates that there is a conserved structural motif that might reflect a common substrate binding fold at the active center of these two classes of enzyme.It is generally believed that enzymes have evolved to catalyze reactions by optimal mechanisms. With the exception of the four classes of proteinases (1) and the two classes of aldolases (2), examples of mechanistically different pairs of enzymes that catalyze the same reaction are very rare. This unusual situation is found with the two classes of dehydroquinase that catalyze the dehydration of 3-dehydroquinate to form 3-dehydroshikimate (3). The reaction is common to two metabolic pathways: the biosynthetic shikimate pathway that is used for the synthesis of aromatic compounds in plants and micro-organisms (4) and the catabolic quinate pathway that enables fungi and some other micro-organism to use quinate as a carbon and energy source (5). The type I dehydroquinases (DHQase) 1 catalyze a cis elimination and are only involved in the biosynthesis of shikimate (6), whereas the type II enzymes, which catalyze a trans elimination (1), have been found to have either a biosynthetic (7) or a catabolic role (8) and in at least one species a dual role (9). Besides being mechanistically distinct, the two classes of DHQase have very different biophysical properties and are apparently unrelated at the level of primary structure (10, 11). The type I enzymes are dimers with a molecular mass of about 46,000 Da; they are heat-labile and use a mechanism that involves the formation of a Schiff-base intermediate followed by the abstraction of a proton by a general base (12-15). In the case of the type I Escherichia coli enzyme the lysine residue has been located (Lys-170) by trapping the Schiff-base intermediate by borohydride reduction (12, 13), and this residue is conserved in all type I sequences (12) as is 16) which is the general base. Both have also been identified as active site residues although their role in substrate binding or catal...

Specificity of substrate recognition by type II dehydroquinases as revealed by binding of polyanions1

“…Therefore we would expect that inhibition would not be as strong as that seen in the S. coelicolor enzyme. The presence of a second sulphate interacting with residues in part of the lid domain, in particular Arg19, a residue already shown to be important for catalysis [8], may help to explain the increase in V max seen in the presence of phosphate. This would suggest that ordering or modification of the conformation of residues in the lid domain of MTDHQase is important for increased catalytic activity.…”

“…The type II DHQases which have been kinetically characterised appear to fall into two groups. The enzymes from organisms such as S. coelicolor and Aspergillus nidulans have high relatively values of k cat , in the range 100–1000 s −1 at pH 7.0 and 25°C [2,8]. By contrast the enzymes from Helicobacter pylori , M. tuberculosis and Neurospora crassa have much lower values of k cat in the range 10 s −1 or lower [3,9–11].…”

Approaches to the Characterisation of Tertiary and Supramolecular Protein Structures by Combination of Protein Chemistry and Mass Spectrometry

“…Beyond the applications to primary structure analysis, recent approaches to the characterisation of higher-order (tertiary) structures, to structure-function studies and even specific non-covalent interactions of proteins are presently emerging as exciting new areas of mass spectrometry [3,5,11]; the recent discovery of ESI-MS as a tool for the direct analysis of supramolecular complexes and molecular recognition processes representing a major theme for this NATO conference [12,13]. Furthermore, the feasibility of soft-ionisation-MS methods to the analysis of multicomponent proteolytic mixtures (peptide mapping) has been demonstrated in several applications, and has been applied successfully to the molecular characterisation of chemical modification sites in proteins [14][15][16][17]. Since the initial studies employing the combination of specific protein-chemical modification reactions and mass spectrometric peptide mapping [3,14], useful structural information has been obtained already, e.g.…”

Mycobacterium tuberculosis Shikimate Pathway Enzymes as Targets for the Rational Design of Anti-Tuberculosis Drugs

“…Tyr24 (M. tuberculosis numbering) is conserved amongst type II enzymes and is proposed to be the base responsible for the extraction of the axial pro-S hydrogen [61,73]. Site-directed mutagenesis experiments showed that Arg23 (S. coelicolor numbering, equivalent to Arg19 in MtDHQase) is an essential amino acid for catalysis [74], although its role is not totally clear as it is present in a flexible loop composed by residues 19-24 (MtDHQase numbering) and it seems to approximate Tyr24 after substrate-binding, lowering its pK a and starting the reaction [75]. It is worth noting that MtDHQase classification as type II is based on structural and theoretical predictions.…”