Structural Interactions between Horseradish Peroxidase C and the Substrate Benzhydroxamic Acid Determined by X-ray Crystallography<sup>,</sup>

Henriksen, A.; Schüller, David; Meno, K.; Welinder, Karen G.; Smith, Andrew T.; Gajhede, Michael

doi:10.1021/bi980234j

Cited by 211 publications

(231 citation statements)

References 47 publications

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“…Computational analyses of the mtCP structure identified an energetically favorable binding site for INH near the ␦-meso edge of the heme (2). This location is similar to that observed for other small aromatic compounds in the crystal structures of class I, II, and III peroxidases (13)(14)(15)(16)). An alternative binding site has also been suggested based upon crystallographic studies of the Burkholderia pseudomallei CP, which places INH in a surface loop structure (17) ϳ10 Å away from the binding site of small aromatic compounds located near the ␦-meso edge of the heme.…”

supporting

confidence: 67%

“…In the HRPC⅐INH and HRPC-cyanide-INH models the hydrazide moiety of INH forms hydrogen bonds to a backbone carbonyl oxygen, INH N3-Pro-139 O (2.7-2.8 Å), and to the distal cata- (Fig. 6B) the INH carbonyl oxygen also forms a hydrogen bond (2.7 Å) to the conserved water located directly above heme iron in the distal pocket but not to the side chain of Arg-38, as would be predicted from the HRPC-BHA crystal structure (16). This is probably due to the high mobility observed for this group in the molecular dynamic simulations.…”

Section: Resultsmentioning

confidence: 68%

“…1B and Fig. 6C) (16). Comparison of the crystal structure of HRPC⅐BHA (16) and the NMR-based model of HRPC⅐INH demonstrates that both BHA and INH can access a similar but not identical set of interactions that may relate to their differing affinities (Fig.…”

Section: Resultsmentioning

confidence: 94%

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Enzyme-catalyzed Mechanism of Isoniazid Activation in Class I and Class III Peroxidases

Pierattelli

Banci

Eady

et al. 2004

Journal of Biological Chemistry

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There is an urgent need to understand the mechanism of activation of the frontline anti-tuberculosis drug isoniazid by the Mycobacterium tuberculosis catalase-peroxidase. To address this, a combination of NMR spectroscopic, biochemical, and computational methods have been used to obtain a model of the frontline anti-tuberculosis drug isoniazid bound to the active site of the class III peroxidase, horseradish peroxidase C. This information has been used in combination with the new crystal structure of the M. tuberculosis catalase-peroxidase to predict the mode of INH binding across the class I heme peroxidase family. An enzyme-catalyzed mechanism for INH activation is proposed that brings together structural, functional, and spectroscopic data from a variety of sources. Collectively, the information not only provides a molecular basis for understanding INH activation by the M. tuberculosis catalase-peroxidase but also establishes a new conceptual framework for testing hypotheses regarding the enzyme-catalyzed turnover of this compound in a number of heme peroxidases.

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supporting

confidence: 67%

Section: Resultsmentioning

confidence: 68%

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Enzyme-catalyzed Mechanism of Isoniazid Activation in Class I and Class III Peroxidases

Pierattelli

Banci

Eady

et al. 2004

Journal of Biological Chemistry

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“…HRP oxidizes both organic and inorganic compounds in the presence of hydrogen peroxide (H 2 O 2 ) as an oxidizing agent (28)(29)(30). We have used Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) as nonfluorescent substrate, which is converted into highly fluorescent resorufin by HRP in the presence of H 2 O 2 ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Probing conformational dynamics of an enzymatic active site by an in situ single fluorogenic probe under piconewton force manipulation

Pal

2016

Proc. Natl. Acad. Sci. U.S.A.

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Unraveling the conformational details of an enzyme during the essential steps of a catalytic reaction (i.e., enzyme-substrate interaction, enzyme-substrate active complex formation, nascent product formation, and product release) is challenging due to the transient nature of intermediate conformational states, conformational fluctuations, and the associated complex dynamics. Here we report our study on the conformational dynamics of horseradish peroxidase using single-molecule multiparameter photon time-stamping spectroscopy with mechanical force manipulation, a newly developed single-molecule fluorescence imaging magnetic tweezers nanoscopic approach. A nascent-formed fluorogenic product molecule serves as a probe, perfectly fitting in the enzymatic reaction active site for probing the enzymatic conformational dynamics. Interestingly, the product releasing dynamics shows the complex conformational behavior with multiple product releasing pathways. However, under magnetic force manipulation, the complex nature of the multiple product releasing pathways disappears and more simplistic conformations of the active site are populated.enzymatic conformational dynamics | magnetic tweezers | mechanical force manipulation | enzymatic product releasing | fluorogenic substrate E nzymes are capable of enhancing biological reaction activity by millions or even billions of times (1). A typical enzymatic turnover cycle involves multiple steps: substrate (S) binding to the active site of the enzyme (E) in forming an enzyme-substrate complex E+S→[E · S]; enzymatic reaction converting substrate to nascent product (P), [E · S]→[E · P]; and product releasing from the enzyme active site, [E · P]→E+P, which completes an enzymatic reaction turnover (2-4). In recent years, it has been widely identified that conformational dynamics plays a crucial role in regulating enzymatic reactions (2, 4-7). The advent of sitespecific mutagenesis, single-molecule (SM) spectroscopic technique, and molecular dynamics simulations have demonstrated complex dynamics involving multiple conformational states during a catalytic cycle (4, 5, 7-13). Fundamentally, not only the enzymatic active site conformational dynamics but also the overall conformational fluctuation dynamics of the protein matrix, providing the required entropy, enthalpy, and corresponding energy landscape, has a profound impact on an enzyme to ultimately possess the power of enhancing reaction rates. Nevertheless, capturing an individual snapshot of each intermediate conformational step remains challenging, mainly due to the transient nature of those intermediate structures, inadequacy of the conventional structure analysis methods to seize those fluctuating structures, and lack of molecular probes that can specifically report the active site environment at each step of an enzymatic reaction. More often than not, the rate-limiting step is the product release from the active site (14). However, a thorough and comprehensive picture of the product release mechanism is still insufficient, mos...

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“…The BHA molecule is a useful probe of the distal heme cavity as it enters into a number of hydrogen bond interactions with Arg 38 , His 42 , Pro 139 (32), and a distal water molecule coordinated to the heme iron, resulting in a 6-c QS complex (33,34). The electronic absorption spectra of the native and Ca 2ϩ -depleted HRPC in the presence of saturating amounts of BHA are very similar (data not shown).…”

Section: Carbon Monoxide and Bha Binding To Camentioning

confidence: 94%

The Critical Role of the Proximal Calcium Ion in the Structural Properties of Horseradish Peroxidase

Howes

Feis

Raimondi³

et al. 2001

Journal of Biological Chemistry

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The extent to which the structural Ca 2؉ ions of horseradish peroxidase (HRPC) are a determinant in defining the heme pocket architecture is investigated by electronic absorption and resonance Raman spectroscopy upon removal of one Ca 2؉ ion. The Fe(III) heme states are modified upon Ca 2؉ depletion, with an uncommon quantum mechanically mixed spin state becoming the dominant species. Ca 2؉ -depleted HRPC forms complexes with benzohydroxamic acid and CO which display spectra very similar to those of native HRPC, indicating that any changes to the distal cavity structural properties upon Ca 2؉ depletion are easily reversed. Contrary to the native protein, the Ca 2؉ -depleted ferrous form displays a low-spin bis-histidyl heme state and a small proportion of high-spin heme. Furthermore, the (Fe-Im) stretching mode downshifts 27 cm ؊1 upon Ca 2؉ depletion revealing a significant structural perturbation of the proximal cavity near the histidine ligand. The specific activity of the Ca 2؉ -depleted enzyme is 50% that of the native form. The effects on enzyme activity and spectral features observed upon Ca 2؉ depletion are reversible upon reconstitution. Evaluation of the present and previous data firmly favors the proximal Ca 2؉ ion as that which is lost upon Ca 2؉ depletion and which likely plays the more critical role in regulating the heme pocket structural and catalytic properties.Peroxidases of the plant peroxidase superfamily are hemecontaining enzymes that oxidize a variety of aromatic molecules in the presence of hydrogen peroxide. They include peroxidases of plant, fungal, and prokaryotic origins that can be divided into three classes based on sequence alignment (1).Additional support for such a classification was gained as the crystal structures of representative enzymes of each class became available. Class I contains bacterial peroxidases and peroxidases from plant mitochondria and chloroplasts, for example most ascorbate peroxidases and cytochrome c peroxidase. Class II contains extracellular fungal peroxidases such as Coprinus cinereus peroxidase (a peroxidase essentially identical to Arthromyces ramosus peroxidase) and lignin-degrading peroxidases. Class III contains secretory plant peroxidases, typified by the classical horseradish peroxidase isoenzyme C (HRPC). The peroxidases of classes II and III share a number of structural elements considered to be of importance for maintaining protein stability and activity. These include calciumbinding sites proximal and distal to the heme and four disulfide bridges (1-5); the latter was in different locations in class II with respect to class III peroxidases. These features are absent in class I peroxidases. Class III peroxidases also have a loop insertion in the sequence, composed of the DЈ, FЈ, and FЉ helices, not found in the other classes. The relationship between calcium binding and enzyme inactivation has been treated primarily by studies on lignin peroxidase (LIP) (6, 7), manganese peroxidase (MNP) (8, 9), and HRPC (10, 11). Thermal (6) and alkaline (7) i...

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Structural Interactions between Horseradish Peroxidase C and the Substrate Benzhydroxamic Acid Determined by X-ray Crystallography^,

Cited by 211 publications

References 47 publications

Enzyme-catalyzed Mechanism of Isoniazid Activation in Class I and Class III Peroxidases

Enzyme-catalyzed Mechanism of Isoniazid Activation in Class I and Class III Peroxidases

Probing conformational dynamics of an enzymatic active site by an in situ single fluorogenic probe under piconewton force manipulation

The Critical Role of the Proximal Calcium Ion in the Structural Properties of Horseradish Peroxidase

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Structural Interactions between Horseradish Peroxidase C and the Substrate Benzhydroxamic Acid Determined by X-ray Crystallography,

Cited by 211 publications

References 47 publications

Enzyme-catalyzed Mechanism of Isoniazid Activation in Class I and Class III Peroxidases

Enzyme-catalyzed Mechanism of Isoniazid Activation in Class I and Class III Peroxidases

Probing conformational dynamics of an enzymatic active site by an in situ single fluorogenic probe under piconewton force manipulation

The Critical Role of the Proximal Calcium Ion in the Structural Properties of Horseradish Peroxidase

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Product

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Structural Interactions between Horseradish Peroxidase C and the Substrate Benzhydroxamic Acid Determined by X-ray Crystallography^,