Detailed differential scanning calorimetry (DSC), steady-state tryptophan fluorescence and far-UV and visible CD studies, together with enzymatic assays, were carried out to monitor the thermal denaturation of horseradish peroxidase isoenzyme c (HRPc) at pH 3.0. The spectral parameters were complementary to the highly sensitive but integral method of DSC. Thus, changes in far-UV CD corresponded to changes in the overall secondary structure of the enzyme, while that in the Soret region, as well as changes in intrinsic tryptophan fluorescence emission, corresponded to changes in the tertiary structure of the enzyme. The results, supported by data about changes in enzymatic activity with temperature, show that thermally induced transitions for peroxidase are irreversible and strongly dependent upon the scan rate, suggesting that denaturation is under kinetic control. It is shown that the process of HRPc denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic schemewhere 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. On the basis of this model, the parameters of the Arrhenius equation were calculated.Keywords: horeseradish peroxidase; differential scanning calorimetry; intrinsic fluorescence; circular dichroism; irreversible denaturation.Horseradish peroxidase (HRP) belongs to the superfamily of the heme-containing plant peroxidases (EC 1.11.1.7), which has been divided into three classes [1], supported in the first instance by comparison of amino-acid sequence data and confirmed by more recent data on crystal structures [2]. Plant peroxidases, including HRP, comprise class III of the superfamily. Although the function of peroxidases is often seen primarily in terms of the conversion of H 2 O 2 to H 2 O, this should not be allowed to mask their wider participation in other reactions, many of which are biologically significant. Despite the enormous interest in peroxidases owing to their broad practical applications in biotechnology, the data concerning their structural stability are sparse. Although several publications have addressed the thermal stability of peroxidases [3±8], to date the mechanism of the process of thermal denaturation remains unclear. It is known that the biological functions of proteins depend on the correct folding of their native structure and that loss of this folded structure leads to an unfolded, inactive state. Consequently, the study of protein stability is important both from the academic and applied points of view.Factors affecting conformational stability have been studied most intensively in proteins under reversible conditions [9±15]. Nevertheless, it is well known that for different reasons many proteins cannot refold in vitro after denaturation such as proteolytic digestion [16], aggregation, loss of prosthetic group, the cis/trans izomerization of certain proline residues [17,18] or chemical modifications [19]. Generally, the thermal denaturat...
The thermal unfolding and domain structure of myosin subfragment 1 (SI) from rabbit skeletal muscles and their changes induced by nucleotide binding were studied by differential scanning calorimetry. The binding of ADP to S1 practically does not influence the position of the thermal transition (maximum at 47.2 "C), while the binding of the nun-hydrolysable analogue of ATP, adenosine 5'-[/Y~-imido]triphosphate (AdoPP[NH]P) to S1, or trapping of ADP in S1 by orthovanadate (V,), shift the maximum of the heat adsorption curve for S1 up to 53.2 and 56.loC, respectively. Such an increase of S1 thermostability in the complexes S1-AdoPP[NH]P and S1-ADPVi is confirmed by results of turbidity and tryptophan fluorescence measurements. The total heat adsorption curves for S1 and its complexes with nucleotides were decomposed into elementary peaks corresponding to the melting of structural domains in the S1 molecule. Quantitative analysis of the data shows that the domain structure of S1 in the complexes Sl-AdoPP[NH]P and S1-ADP-V, is similar and differs radically from that of nucleotide-free S1 and S1 in the S1-ADP complex. These data are the first direct evidence that the S1 molecule can be in two main conformations which may correspond to different states during the ATP hydrolysis : one of them corresponds to nucleotide-free S1 and to the complex S1-ADP, and the other corresponds to the intermediate complexes S1-ATP and S1-ADP-P,. Surprisingly it turned out that the domain structure of S1 with ADP trapped by p-phenylene-N, N'-dimaleimide @PDM) thiol cross-linking almost does not differ from that of the nucleotide-free S1. This means that pPDM-cross-linked S1 in contrast to S1-AdoPP[NH]P and S1-ADP-V, can not be considered a structural analogue of the intermediate complexes S1-ATP and S1-ADP-P,.Muscle contraction and many other manifestations of biological motility are based on the cyclic interaction of myosin heads with actin, which is accompanied by ATP hydrolysis. During steady-state ATP hydrolysis the myosin head is subjected to conformational changes that can be detected by changcs in ultraviolet absorption Enzyme. Chymotrypsin (EC 3.4.21 .I).is used as a stable analogue of the S1-ATP complex.Complexes of S1 with ADP and orthovanadate (Vi) [S, 61 and S1 cross-linked by p-phenylene-N,N-dimaleimide (pPDM) in the presence of ADP [7] are often used as stable analogues of the S1-ADP-Pi complex. The use of stable analogues allows the structure and properties of S1 in the intermediates of the ATPase reaction to be studied. There are numerous data suggesting that the stable analogues of the S1-ATP and S1-ADP-Pi intermediates are similar in structure, but differ from nucleotide-free S1 and S1-ADP [8-201, It should be noted that all these data are in fact indirect since they are based on either studies of the actinbinding properties of S1 [8 -151 or studies of local conformational changes in S1 [16-201. In order to obtain direct proof of the assumption of two main structural states of S1, one must know the effects of nucleoti...
Royal palm tree peroxidase (RPTP) is a very stable enzyme in regards to acidity, temperature, H(2)O(2), and organic solvents. Thus, RPTP is a promising candidate for developing H(2)O(2)-sensitive biosensors for diverse applications in industry and analytical chemistry. RPTP belongs to the family of class III secretory plant peroxidases, which include horseradish peroxidase isozyme C, soybean and peanut peroxidases. Here we report the X-ray structure of native RPTP isolated from royal palm tree (Roystonea regia) refined to a resolution of 1.85A. RPTP has the same overall folding pattern of the plant peroxidase superfamily, and it contains one heme group and two calcium-binding sites in similar locations. The three-dimensional structure of RPTP was solved for a hydroperoxide complex state, and it revealed a bound 2-(N-morpholino) ethanesulfonic acid molecule (MES) positioned at a putative substrate-binding secondary site. Nine N-glycosylation sites are clearly defined in the RPTP electron-density maps, revealing for the first time conformations of the glycan chains of this highly glycosylated enzyme. Furthermore, statistical coupling analysis (SCA) of the plant peroxidase superfamily was performed. This sequence-based method identified a set of evolutionarily conserved sites that mapped to regions surrounding the heme prosthetic group. The SCA matrix also predicted a set of energetically coupled residues that are involved in the maintenance of the structural folding of plant peroxidases. The combination of crystallographic data and SCA analysis provides information about the key structural elements that could contribute to explaining the unique stability of RPTP.
Thermal denaturation of Torpedo culifornicu acetylcholinesterase, a disulfide-linked homodimer with 537 amino acids in each subunit, was studied by differential scanning calorimetry. It displays a single calorimetric peak that is completely irreversible, the shape and temperature maximum depending on the scan rate. Thus, thermal denaturation of acetylcholinesterase is an irreversible process, under kinetic control, which is described well by the twostate kinetic scheme N 5 D, with activation energy 131 * 8 kcal/mol. Analysis of the kinetics of denaturation in the thermal transition temperature range, by monitoring loss of enzymic activity, yields activation energy of 121 t 20 kcal/mol, similar to the value obtained by differential scanning calorimetry. Thermally denatured acetylcholinesterase displays spectroscopic characteristics typical of a molten globule state, similar to those of partially unfolded enzyme obtained by modification with thiol-specific reagents. Evidence is presented that the partially unfolded states produced by the two different treatments are thermodynamically favored relative to the native state.Keywords: acetylcholinesterase; differential scanning calorimetry; irreversible denaturation; molten globule; thioldisulfide exchange; two-state kinetic model Folding of many small globular proteins is a cooperative process in the course of which only two states, the fully unfolded state, U, and the native state, N, are significantly populated. In the case of large proteins, which are generally believed to contain several domains (Jaenicke, 1991;Garel, 1992), folding has generally been considered to be cooperative within each domain, the only species populated in the course of either folding or unfolding being combinations of completely folded and completely unfolded domains (Privalov, 1982;Brandts et al., 1989;Garel, 1992). Evidence is accumulating, however, that another state, intermediate between N and U, can exist. This is a compact state that lacks the unique tertiary structure of the native protein but possesses substantial secondary structure. This state has been named the molten globule (MG) state (Kuwajima, 1989;Kim & Baldwin, 1990;Ptitsyn, 1992). The MG state is considered to serve as an intermediate on the pathway from the nascent polypeptide chain to the fully folded native protein in vivo (Gething & Sambrook, 1992). For most proteins, the MG state is unstable under physiological conditions and readily converts to the N state in vitro (Ptitsyn, 1992 Because the definition of the MG state is controversial (see, for example, Griko et al., 1994; Ewbank et al., 1995;Okazaki et al., 1995), in the present paper this term is used to refer to compact states of acetylcholinesterase (AChE) that preserve substantial secondary structure and lack most of the tertiary structure of the native enzyme. We have shown recently that exposure of a native dimeric form of Torpedo AChE to various treatments generates long-lived partially unfolded species displaying many of the characteristics of the MG...
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