An asynchronous unfolding among molecular different regions of lobster D-glyceraldehyde-3-phosphate dehydrogenase and maltotetraose-forming amylase from an Alcaligenes sp. during guanidine denaturation.
Changes in ultraviolet absorbance and intrinsic protein fluorescence of 1,4-alpha-D-glucan maltotetrahydrolase (EC 3.2.1.60) from an Alcaligenes sp. (Gram-negative bacteria 537.1) and D-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) have been compared with their inactivation during denaturation in guanidinium-Cl solutions. The two enzymes were completely inactivated at GuHCl concentrations less than 0.6 M and this was accompanied by marked absorbance and intrinsic fluorescence changes suggesting exposure of aromatic residues. The changes of the intrinsic fluorescence of the amylase have a relatively constant plateau in emission intensities and maxima at GuHCl concentrations from 0.8-2.0 M, similar to that of muscle GAPDH. The relative activity of the enzyme increased markedly in dilute GuHCl solutions accompanied by very little change of its intrinsic fluorescence at 8 degrees C. The kinetic decrease in emission intensities, excited respectively by 230 nm and 292 nm, was different for the two enzymes. The inactivation was a biphasic process with a fast phase faster than the unfolding rate as measured by fluorescence changes in 0.5 M GuHCl solution. Similar to the inactivation process, changes in intensity of 410 nm NAD fluorescent derivative of GAPDH which is in situ at the active site is also a biphasic process under the same condition. It appears that there may be an unfolding intermediate state of the enzymes and an asynchronous unfolding process among the different regions in the molecules during GuHCl denaturation, this may be due to differences in their flexibility.
National Laboratory of Biomacromolecules, Academia Sinica, Beijing, China.
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D000421:Alcaligenes / Q000201:enzymology; D000818:Animals; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D008320:Maltose / Q000031:analogs & derivatives / Q000096:biosynthesis; D008121:Nephropidae / Q000201:enzymology; D009844:Oligosaccharides / Q000096:biosynthesis; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D000516:alpha-Amylases / Q000737:chemistry* / Q000378:metabolism
D006146:Guanidines; D009844:Oligosaccharides; D008320:Maltose; C009819:maltotetraose; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D000516:alpha-Amylases; D019791:Guanidine