Horseradish peroxidase: a modern view of a classic enzyme

Veitch, Nigel C.

doi:10.1016/j.phytochem.2003.10.022

Cited by 1,181 publications

(903 citation statements)

References 41 publications

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“…HRP is a 44-kDa monomeric metalloenzyme containing a heme prosthetic group and each substrate turnover is produced from a single catalytic cycle (27,28). HRP oxidizes both organic and inorganic compounds in the presence of hydrogen peroxide (H 2 O 2 ) as an oxidizing agent (28)(29)(30).…”

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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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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“…189 SBP has advantages over HRP in terms of catalytic activity and stability [55]; these 190 can be exploited in biosensors. Also, unlike HRP, SBP is active in the pH range [2][3][4][5][6]191 offering a greater range of potential biosensing applications [56]. The first SBP 192 biosensor was reported in 1995 by Vreeke and co-workers [57] as a thermostable 193 wired enzyme electrode using an osmium-based mediator, which aids electron transfer 194 from the active site to the electrode, modified by an epoxide.…”

Section: Sbp In Biosensors 188mentioning

confidence: 99%

Horseradish and soybean peroxidases: comparable tools for alternative niches?

Ryan

Carolan

Ó’Fágáin

2006

Trends in Biotechnology

141

102

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Horseradish and soybean peroxidases (HRP and SBP, respectively) are useful 9 biotechnological tools. HRP is often termed the classical plant heme peroxidase, and 10 although it has been studied for decades our understanding has deepened since its 11 cloning and subsequent expression, which has enabled numerous mutational and 12 protein engineering studies. SBP, however, has been neglected until recently; despite 13 offering a real alternative to HRP that actually outperforms it in terms of stability. 14 SBP is now used in numerous biotechnological applications, including biosensors. 15Review of both is timely. This article summarises and discusses the main insights into 16 the structure and mechanism of HRP, with special emphasis on HRP mutagenesis, and 17 outlines its use in a variety of applications. It also reviews current knowledge and 18 applications to date of SBP, particularly biosensors. The final paragraphs speculate on 19 the future of plant heme-based peroxidases, with probable trends outlined and 20 explored. 21 22

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“…Applications of horseradish peroxidase (HRP) as noted Hollmann and Arends [1] include removal of peroxide, phenols, amines, indols and other heterocyclic compounds from materials such as food stuffs and industrial waste, as oil refineries, plastics, resins, textiles, iron and steel, forestry industries waste water containing phenolic compounds, synthesis of various aromatic chemicals, in biocatalysis and in radical polymerization. Veitch [2] have reported the decolorization and removal of textile dyes from polluted water and dyeing effluents by using soluble and immobilized peroxidases. Azevedo et al [3] note that about 82% of the commercially available HRP is used in kits to test for levels of glucose, uric acid, cholesterol and lactose.…”

Section: Introductionmentioning

confidence: 99%

Biocatalytic properties of horseradish root extract peroxidase (HRP)

Purev¹,

J²,

Tsevelmaa³

et al. 2014

Mong. J. Chem.

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Abstract:The objective of the present work was to examine biocatalytic properties of peroxidase in horseradish acclimatized in our country. We have found that horseradish root extract's peroxidase (HRP) has K M 2.5 mM and V max 5.36 mM·s -1 . Maximum activity (pH opt ) was estimated at pH 6.0 and enzyme is more stable in alkali, than in acid. The optimum temperature (T opt ) for HRP is 40 o C and the enzyme is not stable to temperature influence. The horseradish root's extract retains enzymatic activity within 21 days.

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Horseradish peroxidase: a modern view of a classic enzyme

Cited by 1,181 publications

References 41 publications

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

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

Horseradish and soybean peroxidases: comparable tools for alternative niches?

Biocatalytic properties of horseradish root extract peroxidase (HRP)

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