Aspartate-induced aminoacylase folding and forming of molten globule.
Aspartate is an osmolyte found in some marine invertebrates and cyclostome fish. The aspartate-induced unfolding of N-acylamino acid amido hydrolase (aminoacylase) has been studied by measuring enzyme activity, fluorescence emission spectra, 8-anilino-1-naphthalenesulfonate (ANS) fluorescence spectra and far-UV circular dichroism (CD) spectra. The results showed that aspartate caused the inactivation and unfolding of aminoacylase. Surprisingly, increasing concentration of aspartate showed the "acid-induced folding", which used to be seen only in strong acids or salts at much lower pH. Although aspartate has the pI of 2.77 that is the lowest among all the free amino acids, it is actually a weak acid. It is thus of great interest why it causes this phenomenon to happen. The relative change of intrinsic fluorescence and ANS binding spectra have shown that there existed a stable molten globule state of aminoacylase with slightly disrupted tertiary structure and more hydrophobic surface. The molten globule state indicates that intermediates existed during aminoacylase refolding process. Unlike the other acids, such as trichloroacetic acid, there is no precipitation observed as the aspartate concentrations increased. It suggests the aspartate anions have an osmotic effect for the molten globule formed during unfolding process. Binding of aspartate anion to the protonated protein, which minimizes the intramolecular repulsion, might explain the osmotic effect of this amino acid in the nature. The results also showed the Apo-aminoacylase followed similar rules as Holo-enzyme, which suggested the zinc ion may play more important roles on activity other than structure.
Xie
Qiang
Q
Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, PR China.
Guo
Tai
T
Wang
Tingting
T
Lu
Jie
J
Zhou
Hai-Meng
HM
D000581:Amidohydrolases / Q000737:chemistry* / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry / Q000494:pharmacology*; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D012492:Salts / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013552:Swine
D000817:Anilino Naphthalenesulfonates; D001059:Apoproteins; D012492:Salts; D001224:Aspartic Acid; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D000581:Amidohydrolases; C019672:aminoacylase I