Dissociation, unfolding and refolding trials of pig kidney 3,4-dihydroxyphenylalanine (dopa) decarboxylase.
The effect of guanidinium chloride (GuCl) on enzyme activity, hydrodynamic volume, circular dichroism, and fluorescence of 3,4-dihydroxyphenylalanine (Dopa) decarboxylase from pig kidney (pkDDC) was studied under equilibrium conditions. Unfolding proceeds in at least three stages. The first transition, occurring between 0 and 1 M GuCl, gives rise to a dimeric inactive species which has lost pyridoxal 5'-phosphate (PLP), and has a high tendency to aggregate, but retains almost all of the native spectroscopic characteristics. The second equilibrium transition, between 1 and 2.2 M GuCl, involves dimer dissociation, with some loss of tertiary and secondary structure. Additionally, gross conformational changes at or near the PLP microenvironment were detected by fluorescence of NaBH4-reduced enzyme. The third step, presumably representing complete unfolding of pkDDC, appears to be complete at 4.5 M GuCl, as indicated by the lack of further substantial changes in any of the signals being studied. Attempts at refolding resulted in the findings that: (1) partial reactivation is observed only starting from enzyme denatured at concentrations below 1.5 M GuCl, and (2) starting from completely denatured protein, the refolding process is apparently reversible down to concentrations of approx. 2 M GuCl. Taken together, this would seem to indicate that the monomer-dimer transition is impaired under the experimental conditions tested. A plausible model is presented for the unfolding/refolding of pkDDC.
Dominici
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P
Università Cattolica del Sacro Cuore, Facoltà di Medicina e Chirurgia A.Gemelli, Rome, Italy.
Moore
P S
PS
Borri Voltattorni
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C
D000818:Animals; D002942:Circular Dichroism; D003067:Coenzymes / Q000378:metabolism; D004296:Dopa Decarboxylase / Q000737:chemistry* / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology*; D009391:Nephelometry and Turbidimetry; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013552:Swine
D003067:Coenzymes; D006146:Guanidines; D004296:Dopa Decarboxylase; D019791:Guanidine