Stereochemical Evidence for Decomposition of Reactive Intermediates by Active Site Water in the Metabolism of 8-Alkyl Xanthines by P450 1A2

1998 ◽  
Vol 120 (21) ◽  
pp. 5337-5338 ◽  
Author(s):  
Jagdish K. Racha ◽  
Kent L. Kunze
Keyword(s):  
Active Site ◽  
Reactive Intermediates ◽  
Site Water

scholarly journals Relebactam Is a Potent Inhibitor of the KPC-2 β-Lactamase and Restores Imipenem Susceptibility in KPC-Producing Enterobacteriaceae

2018 ◽  
Vol 62 (6) ◽  
Author(s):  
Krisztina M. Papp-Wallace ◽  
Melissa D. Barnes ◽  
Jim Alsop ◽  
Magdalena A. Taracila ◽  
Christopher R. Bethel ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Rate Constant ◽  
Active Site ◽  
Klebsiella Oxytoca ◽  
Enterobacter Aerogenes ◽  
Clinical Isolates ◽  
Piperidine Ring ◽  
Water Molecules ◽  
Content Type ◽  
Site Water

ABSTRACT The imipenem-relebactam combination is in development as a potential treatment regimen for infections caused by Enterobacteriaceae possessing complex β-lactamase backgrounds. Relebactam is a β-lactamase inhibitor that possesses the diazabicyclooctane core, as in avibactam; however, the R1 side chain of relebactam also includes a piperidine ring, whereas that of avibactam is a carboxyamide. Here, we investigated the inactivation of the Klebsiella pneumoniae carbapenemase KPC-2, the most widespread class A carbapenemase, by relebactam and performed susceptibility testing with imipenem-relebactam using KPC-producing clinical isolates of Enterobacteriaceae . MIC measurements using agar dilution methods revealed that all 101 clinical isolates of KPC-producing Enterobacteriaceae ( K. pneumoniae , Klebsiella oxytoca , Enterobacter cloacae , Enterobacter aerogenes , Citrobacter freundii , Citrobacter koseri , and Escherichia coli ) were highly susceptible to imipenem-relebactam (MICs ≤ 2 mg/liter). Relebactam inhibited KPC-2 with a second-order onset of acylation rate constant ( k 2 / K ) value of 24,750 M −1 s −1 and demonstrated a slow off-rate constant ( k off ) of 0.0002 s −1 . Biochemical analysis using time-based mass spectrometry to map intermediates revealed that the KPC-2–relebactam acyl-enzyme complex was stable for up to 24 h. Importantly, desulfation of relebactam was not observed using mass spectrometry. Desulfation and subsequent deacylation have been observed during the reaction of KPC-2 with avibactam. Upon molecular dynamics simulations of relebactam in the KPC-2 active site, we found that the positioning of active-site water molecules is less favorable for desulfation in the KPC-2 active site than it is in the KPC-2–avibactam complex. In the acyl complexes, the water molecules are within 2.5 to 3 Å of the avibactam sulfate; however, they are more than 5 to 6 Å from the relebactam sulfate. As a result, we propose that the KPC-2–relebactam acyl complex is more stable than the KPC-2–avibactam complex. The clinical implications of this difference are not currently known.

How an Enzyme Tames Reactive Intermediates:  Positioning of the Active-Site Components of Lysine 2,3-Aminomutase during Enzymatic Turnover As Determined by ENDOR Spectroscopy

2006 ◽  
Vol 128 (31) ◽  
pp. 10145-10154 ◽  
Author(s):  
Nicholas S. Lees ◽  
Dawei Chen ◽  
Charles J. Walsby ◽  
Elham Behshad ◽  
Perry A. Frey ◽  
...  
Keyword(s):  
Active Site ◽  
Reactive Intermediates ◽  
Endor Spectroscopy

scholarly journals The nature of chemical innovation: new enzymes by evolution

2015 ◽  
Vol 48 (4) ◽  
pp. 404-410 ◽  
Author(s):  
Frances H. Arnold
Keyword(s):  
Cytochrome P450 ◽  
Active Site ◽  
Reactive Intermediates ◽  
Transfer Reactions ◽  
Amino Acid Substitutions ◽  
Chemical Catalysis ◽  
Nitrene Transfer ◽  
Enzyme Systems ◽  
Natural Enzyme ◽  
And Function

AbstractI describe how we direct the evolution of non-natural enzyme activities, using chemical intuition and information on structure and mechanism to guide us to the most promising reaction/enzyme systems. With synthetic reagents to generate new reactive intermediates and just a few amino acid substitutions to tune the active site, a cytochrome P450 can catalyze a variety of carbene and nitrene transfer reactions. The cyclopropanation, N–H insertion, C–H amination, sulfimidation, and aziridination reactions now demonstrated are all well known in chemical catalysis but have no counterparts in nature. The new enzymes are fully genetically encoded, assemble and function inside of cells, and can be optimized for different substrates, activities, and selectivities. We are learning how to use nature's innovation mechanisms to marry some of the synthetic chemists’ favorite transformations with the exquisite selectivity and tunability of enzymes.

scholarly journals Probing the Role of Active Site Water in the Sesquiterpene Cyclization Reaction Catalyzed by Aristolochene Synthase

Biochemistry ◽  
2016 ◽  
Vol 55 (20) ◽  
pp. 2864-2874 ◽  
Author(s):  
Mengbin Chen ◽  
Wayne K. W. Chou ◽  
Naeemah Al-Lami ◽  
Juan A. Faraldos ◽  
Rudolf K. Allemann ◽  
...  
Keyword(s):  
Active Site ◽  
Cyclization Reaction ◽  
Aristolochene Synthase ◽  
Site Water

scholarly journals Potent Inhibitors of a Shikimate Pathway Enzyme from Mycobacterium tuberculosis

2011 ◽  
Vol 286 (18) ◽  
pp. 16197-16207 ◽  
Author(s):  
Sebastian Reichau ◽  
Wanting Jiao ◽  
Scott R. Walker ◽  
Richard D. Hutton ◽  
Edward N. Baker ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Conformational Changes ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Condensation Reaction ◽  
Shikimate Pathway ◽  
Enzyme Reaction ◽  
Multidrug Resistant ◽  
Active Site Residues ◽  
Site Water

Tuberculosis remains a serious global health threat, with the emergence of multidrug-resistant strains highlighting the urgent need for novel antituberculosis drugs. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the shikimate pathway for the biosynthesis of aromatic compounds. This pathway has been shown to be essential in Mycobacterium tuberculosis, the pathogen responsible for tuberculosis. DAH7PS catalyzes a condensation reaction between P-enolpyruvate and erythrose 4-phosphate to give 3-deoxy-d-arabino-heptulosonate 7-phosphate. The enzyme reaction mechanism is proposed to include a tetrahedral intermediate, which is formed by attack of an active site water on the central carbon of P-enolpyruvate during the course of the reaction. Molecular modeling of this intermediate into the active site reported in this study shows a configurational preference consistent with water attack from the re face of P-enolpyruvate. Based on this model, we designed and synthesized an inhibitor of DAH7PS that mimics this reaction intermediate. Both enantiomers of this intermediate mimic were potent inhibitors of M. tuberculosis DAH7PS, with inhibitory constants in the nanomolar range. The crystal structure of the DAH7PS-inhibitor complex was solved to 2.35 Å. Both the position of the inhibitor and the conformational changes of active site residues observed in this structure correspond closely to the predictions from the intermediate modeling. This structure also identifies a water molecule that is located in the appropriate position to attack the re face of P-enolpyruvate during the course of the reaction, allowing the catalytic mechanism for this enzyme to be clearly defined.

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