Stabilization of the shikimate pathway enzyme dehydroquinase by covalently bound ligand

Kleanthous, Colin; Reilly, M.; Cooper, Alan; Kelly, Sharon M.; Price, Nicholas C.; Coggins, John R.

doi:10.1016/s0021-9258(18)99103-9

Cited by 29 publications

(3 citation statements)

References 24 publications

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“…12 The Lys170 residue is conserved in all the known type I DHQDs [13][14][15][16] and has been identified to form the covalent Schiff base intermediates with the substrate by both chemical modification and peptide mapping studies. The reduction of the intermediates significantly affects the stability of the resulting enzyme, 17,18 which is consistent with the fact that the mutation of K170A results in a ∼10 6 -fold reduction in catalytic activity. 12 Another important residue is His143 as the mutation of H143A also results in a ∼10 6 -fold reduction in catalytic activity and several experimental results imply that this residue has a wide ranging influence on the mechanism of the enzyme.…”

Section: Introductionsupporting

confidence: 77%

New insights into the mechanism of the Schiff base hydrolysis catalyzed by type I dehydroquinate dehydratase from S. enterica: a theoretical study

Yao

2012

Org. Biomol. Chem.

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The reaction pathway of Schiff base hydrolysis catalyzed by type I dehydroquinate dehydratase (DHQD) from S. enterica has been studied by performing molecular dynamics (MD) simulations and density functional theory (DFT) calculations and the corresponding potential energy profile has also been identified. On the basis of the results, the catalytic hydrolysis process for the wild-type enzyme consists of three major reaction steps, including nucleophilic attack on the carbon atom involved in the carbon-nitrogen double bond of the Schiff base intermediate by a water molecule, deprotonation of the His143 residue, and dissociation between the product and the Lys170 residue of the enzyme. The remarkable difference between this and the previously proposed reaction mechanism is that the second step here, absent in the previously proposed reaction mechanism, plays an important role in facilitating the reaction through a key proton transfer by the His143 residue, resulting in a lower energy barrier. Comparison with our recently reported results on the Schiff base formation and dehydration processes clearly shows that the Schiff base hydrolysis is rate-determining in the overall reaction catalyzed by type I DHQD, consistent with the experimental prediction, and the calculated energy barrier of ∼16.0 kcal mol(-1) is in good agreement with the experimentally derived activation free energy of ∼14.3 kcal mol(-1). When the imidazole group of His143 residue is missing, the Schiff base hydrolysis is initiated by a hydroxide ion in the solution, rather than a water molecule, and both the reaction mechanism and the kinetics of Schiff base hydrolysis have been remarkably changed, clearly elucidating the catalytic role of the His143 residue in the reaction. The new mechanistic insights obtained here will be valuable for the rational design of high-activity inhibitors of type I DHQD as non-toxic antimicrobials, anti-fungals, and herbicides.

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Section: Introductionsupporting

confidence: 77%

New insights into the mechanism of the Schiff base hydrolysis catalyzed by type I dehydroquinate dehydratase from S. enterica: a theoretical study

Yao

2012

Org. Biomol. Chem.

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“…3 The biophysical properties of this inactivated enzyme have been compared with those of the native, underivatized enzyme. 4 Using sodium [3H]borohydride a radiolabel was introduced into the enzyme-ligand adduct. Proteolytic degradation and sequencing of the labeled enzyme identified the modified residue as lysine-170 in the Escherichia coli enzyme.5 This residue is strongly conserved in all type I dehydroquinases.6 Such evidence is considered diagnostic of an imine intermediate in a catalytic mechanism despite the fact that it is indirect.…”

Section: T H I S C O N T E N T Imentioning

confidence: 99%

Observation of an imine intermediate on dehydroquinase by electrospray mass spectrometry

Shneier¹,

Kleanthous²,

Deka³

et al. 1991

J. Am. Chem. Soc.

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“…Another example is of dehydroquinase, where a reaction intermediate/transition state analogue was attached by borohydride reduction. This modification markedly stabilized the enzyme to heat, guanidine hydrochloride, and proteolysis (52). Enzyme-substrate intermediates, of β-lactamases, for example, appear to be either stabilized or destabilized with respect to the apoenzyme (33,39); one would expect stabilization by good (specific) substrates.…”

Section: Resultsmentioning

confidence: 99%

Thermodynamic Evaluation of a Covalently Bonded Transition State Analogue Inhibitor: Inhibition of β-Lactamases by Phosphonates

Nagarajan

Pratt

2004

Biochemistry

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Serine beta-lactamases are inhibited by phosphonate monoesters in a reaction that involves phosphonylation of the active site serine residue. This reaction is much more rapid than the hydrolysis of these inhibitors in solution under the same conditions. The beta-lactamase active site therefore must have the ability to stabilize not only the anionic tetrahedral transition states of the acyl transfer reactions of substrates but also the pentacoordinated transition state(s) of phosphyl transfer reactions. A series of p-nitrophenyl arylphosphonates have been synthesized and the rate constants for their inhibition of the class C beta-lactamase of Enterobacter cloacae P99 determined. There is no direct correlation between these rate constants and the dissociation constants of analogous aryl boronic acids, where the latter are believed to generate good tetrahedral transition state analogue structures. Thus, the mode of stabilization of pentacoordinated phosphorus transition states by the beta-lactamase active site is qualitatively different from that of tetrahedral transition states. Molecular modeling suggests that the difference arises from different positioning of the side chain and of one of the oxygen ligands. In principle, the quality of the stable tetrahedral phosphonate complex as a transition state analogue structure can be assessed from the effect of its formation on the stability of the protein. Phosphonylation of the P99 beta-lactamase, however, had little effect on the stability of the protein, as measured both by thermal and guanidine hydrochloride denaturation. Consideration of the results of similar experiments with the Staphylococcus aureus PC1 beta-lactamase, where considerable stabilization is observed in thermal melting and, to a lesser degree, in formation of the molten globule in guanidine hydrochloride, but not in the complete unfolding transition in guanidine, suggests that results from the method may be strongly influenced by the interactions of the ligand with its environment in the unfolded state of the protein. Thus, quantitative estimates of the quality of a covalently bonded transition state analogue cannot generally be achieved by this method.

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Stabilization of the shikimate pathway enzyme dehydroquinase by covalently bound ligand

Cited by 29 publications

References 24 publications

New insights into the mechanism of the Schiff base hydrolysis catalyzed by type I dehydroquinate dehydratase from S. enterica: a theoretical study

New insights into the mechanism of the Schiff base hydrolysis catalyzed by type I dehydroquinate dehydratase from S. enterica: a theoretical study

Observation of an imine intermediate on dehydroquinase by electrospray mass spectrometry

Thermodynamic Evaluation of a Covalently Bonded Transition State Analogue Inhibitor: Inhibition of β-Lactamases by Phosphonates

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