Steady-state kinetics of Roystonea regia palm tree peroxidase

ABSTRAC

Caracterización químico física de la Peroxidasa de Palma Chamaerops excelsa

[ES] El objetivo final de este trabajo es definir una enzima con unas características espectrales mucho mejores que las de la peroxidasa de rábano picante, capaz de sustituirla en la mayor parte de sus aplicaciones biotecnológicas. Una enzima más estable permitiría realizar los procesos más eficazmente y en mejores condiciones de pH y temperatura, lo que sería rentable y, además, beneficiosa para el Medio Ambiente.[EN] The ultimate goal of this work is to define an enzyme with spectral characteristics much better than horseradish peroxidase, capable of replacing it in most of their biotechnological applications. A more stable enzyme would make the process more efficient and better conditions of pH and temperature, which would be economical, and beneficial to the environment

Thermal stability of peroxidase from Chamaerops excelsa palm tree at pH 3

7 pages, 5 figures, 4 tables.-- PMID: 19428462 [PubMed].-- Printed version published May 1, 2009.The structural stability of a peroxidase, a dimeric protein from palm tree Chamaerops excelsa leaves (CEP), has been characterized by high-sensitivity differential scanning calorimetry, circular dichroism and steady-state tryptophan fluorescence at pH 3. The thermally induced denaturation of CEP at this pH value is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Moreover, thermally induced transitions at this pH value are dependent on the protein concentration, leading to the conclusion that in solution CEP behaves as dimer, which undergoes thermal denaturation coupled with dissociation. Analysis of the kinetic parameters of CEP denaturation at pH 3 was accomplished on the basis of the simple kinetic scheme N -(k)-> D [View source], where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state, and thermodynamic information was obtained by extrapolation of the kinetic transition parameters to an infinite heating rate.This work was partially supported by projects SA-06-00-0 ITACYL-Universidad de Salamanca and SA 129A07 (Junta de Castilla y León) and BFU2004-01432 and BFU2007-68107-C02-02/BMC (Ministerio de Educación y Ciencia) Spain. L.S.Z was fellowship holders from the Junta de Castilla y León, Spain (Ref. EDU/1490/2003).Peer reviewe

Substrate specificity of the Chamaerops excelsa palm tree peroxidase. A steady-state kinetic study

The steady state kinetic mechanism of the H2O2-supported oxidation of different organic substrates by peroxidase from leaves of Chamaerops excelsa palm trees (CEP) has been investigated. An analysis of the initial rates vs. H2O2 and reducing substrate concentrations is consistent with a substrate-inhibited Ping-Pong Bi Bi reaction mechanism. The phenomenological approach expresses the peroxidase Ping-Pong mechanism in the form of the Michaelis–Menten equation and leads to an interpretation of the effects in terms of the kinetic parameters K(H2O2, m), K(AH2, m), k(cat), K(H2O2, SI), K(AH2, SI) and of the microscopic rate constants k1 and k3 of the shared three-step catalytic cycle of peroxidases.Funding from Consejeria de Educación (projects SA129A07 and SA052A10-2) and Consejeria de Agricultura y Ganaderia (Project SA06000) of the Regional Government of Castilla and León (Junta de Castilla y León, Spain) is acknowledged.Peer reviewe

Purification and structural stability of white Spanish broom (Cytisus multiflorus) peroxidase

New plant peroxidase has been isolated to homogeneity from the white Spanish broom Cytisus multiflorus. The enzyme purification steps included homogenization, NH4SO4 precipitation, extraction of broom colored compounds and consecutive chromatography on Phenyl-Sepharose, HiTrap™ SP HP and Superdex-75 and 200. The novel peroxidase was characterized as having a molecular weight of 50 ± 3 kDa. Steady-state tryptophan fluorescence and far-UV circular dichroism (CD) studies, together with enzymatic assays, were carried out to monitor the structural stability of C. multiflorus peroxidase (CMP) at pH 7.0. Thus changes in far-UV CD corresponded to changes in the overall secondary structure of enzyme, while changes in intrinsic tryptophan fluorescence emission corresponded to changes in the tertiary structure of the enzyme. It is shown that the process of CMP denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic scheme, N->D , where k is a first-order kinetic constant that changes with temperature following the Arrhenius equation; N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated.This work was partially supported by Projects SA-06-00-0 ITACYL-Universidad de Salamanca, SA 129A07, and SA052A10-2 funded by the Instituto Tecnológico Agrario de Castilla y León and the Consejeria de Educación de la Junta de Castilla y León (Spain).Peer reviewe

Palm peroxidades: the most robust enzymes

Peroxidases are ubiquitous enzymes that catalyze a variety of oxygen-transfer reactions and are thus potentially useful for industrial and biomedical applications. Over the last decade, several studies have shown that peroxidases isolated from the leaves of different kinds of palm trees such as the royal palm (Roystonea regia), the date palm (Phoenix dactylifera), the African oil palm (Elaeis guineensis), the ruffle palm (Aiphanes cariotifolia) and the windmill palms (Trachycarpus fortunei and Chamaerops excelsa) exhibit higher activity and stability than commercially available peroxidases isolated from, for example, horseradish roots (Armoracia rusticana) and soybean (Glycine max). Here, the structure, thermal denaturation, and the catalytic cycle of peroxidases from palm trees are reviewed and compared with those of other plant peroxidases. In addition, we report the biotechnological potential of palm peroxidases and their implications in cellular aging and diseases, such as Refsum’s and Alzheimer’s diseases. This paper summarizes the main characteristics of the palm peroxidases studied.Peer reviewe

Crystal structure analysis of peroxidase from the palm tree Chamaerops excelsa

Palm tree peroxidases are known to be very stable enzymes and the peroxidase from the Chamaerops excelsa (CEP), which has a high pH and thermal stability, is no exception. To date, the structural and molecular events underscoring such biochemical behavior have not been explored in depth. In order to identify the structural characteristics accounting for the high stability of palm tree peroxidases, we solved and refined the X-ray structure of native CEP at a resolution of 2.6 Å. The CEP structure has an overall fold typical of plant peroxidases and confirmed the conservation of characteristic structural elements such as the heme group and calcium ions. At the same time the structure revealed important modifications in the amino acid residues in the vicinity of the exposed heme edge region, involved in substrate binding, that could account for the morphological variations among palm tree peroxidases through the disruption of molecular interactions at the second binding site. These modifications could alleviate the inhibition of enzymatic activity caused by molecular interactions at the latter binding site. Comparing the CEP crystallographic model described here with other publicly available peroxidase structures allowed the identification of a noncovalent homodimer assembly held together by a number of ionic and hydrophobic interactions. We demonstrate, that this dimeric arrangement results in a more stable protein quaternary structure through stabilization of the regions that are highly dynamic in other peroxidases. In addition, we resolved five N-glycosylation sites, which might also contribute to enzyme stability and resistance against proteolytic cleavage