Summary
The guanidinium chloride (GuCI) induced unfolding of dopa decarboxylase (DDC) has been studied by monitoring its effects on enzymatic activity. hydrodynamic volume. far-uv cd. and intrinsic fluorescence. Due to its spectral properties. pyridoxal 5′-phosphate (PLP) was also used as a probe of structural changes occuring at the active site during the unfolding/refolding process. The combination of these studies revealed that the unfolding of DDC occurs as a multi-step process minimally composed of at least three steps that could be identified at equilibrium. The first step occurs at 0–1 M GuCI and gives rise to an inactive dimeric species which is devoid of coenzyme. At 1–2.2 M GuCI a second transition is observed. consisting in subunit dissociation and partial loss of tertiary and secondary structure accompanied by structural alterations of the coenzyme microenvironment leading to a “structured monomer”. The third and final phase of the unfolding process is complete at about 4.5 M GuCl and is characterized by complete subunit unfolding. Further increasing the GuCI concentration failed to cause a change in any of the parameters followed. While enzymatic activity could never be regained starting from fully denatured protein. partial reactivation was seen starting from enzyme treated with GuCI concentrations less than 1.5 M. Together with an more in-depth investigation into the refolding, it is suggested that. at least under the experimental condition tested, the monomer-dimer transition is impaired during this process. These results are discussed in terms of a model for the unfolding/refolding of pig kidney dopa decarboxylase.
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References
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© 1994 Birkhäuser Verlag Basel/Switzerland
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Dominici, P., Moore, P.S., Voltattorni, C.B. (1994). GuCI-induced unfolding of pig kidney dopa decarboxylase: Evidence for a multi-state process. In: Marino, G., Sannia, G., Bossa, F. (eds) Biochemistry of Vitamin B6 and PQQ. Advances in Life Sciences. Birkhäuser Basel. https://doi.org/10.1007/978-3-0348-7393-2_25
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DOI: https://doi.org/10.1007/978-3-0348-7393-2_25
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