title: Conformational and activity changes during guanidine denaturation of D-glyceraldehyde-3-phosphate dehydrogenase.
abstract: Changes in intrinsic protein fluorescence of lobster muscle D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) have been compared with inactivation of the enzyme during denaturation in guanidine solutions. The holoenzyme is completely inactivated at guanidine concentrations less than 0.5 M and this is accompanied by a red shift of the emission maximum at 335 nm and a marked decrease in intensity of the intrinsic fluorescence. At 0.5 M guanidine, the inactivation is a slow process, with a first-order rate constant of 2.4 X 10(-3) s-1. A further red shift in the emission maximum and a decrease in intensity occur at guanidine concentrations higher than 1.5 M. The emission peak at 410 nm of the fluorescent NAD derivative introduced at the active site of this enzyme (Tsou, C.L. et al. (1983) Biochem. Soc. Trans. 11, 425-429) shows both a red shift and a marked decrease in intensity at the same guanidine concentration required to bring about the inactivation and the initial changes in the intrinsic fluorescence of the holoenzyme. It appears that treatment by low guanidine concentrations leads to both complete inactivation and perturbation of the active site conformation and that a tryptophan residue is situated at or near the active site.
mesh_terms: D000818:Animals; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D009243:NAD / Q000378:metabolism; D008121:Nephropidae / Q000201:enzymology; D009994:Osmolar Concentration; D011487:Protein Conformation; D011489:Protein Denaturation; D012996:Solutions; D013050:Spectrometry, Fluorescence
chemical_list: D006146:Guanidines; D012996:Solutions; D009243:NAD; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D019791:Guanidine