Unfolding and inactivation during thermal denaturation of an enzyme that exhibits phytase and acid phosphatase activities.
The thermostability of an enzyme that exhibits phytase and acid phosphatase activities was studied. Kinetics of inactivation and unfolding during thermal denaturation of the enzyme were compared. The loss of phytase activity on thermal denaturation is most suggestive of a reversible process. As for acid phosphatase activities, an interesting phenomenon was observed; there are two phases in thermal inactivation: when the temperature was between 45 and 50 degrees C, the thermal inactivation could be characterized as an irreversible inactivation which had some residual activity and when the temperature was above 55 degrees C, the thermal inactivation could be characterized as an irreversible process which had no residual activity. The microscopic rate constants for the free enzyme and substrate-enzyme complex were determined by Tsou's method [Adv. Enzymol. Relat. Areas Mol. Biol. 61 (1988) 381]. Fluorescence analyses indicate that when the enzyme was treated at temperatures below 60 degrees C for 60 min, the conformation of the enzyme had no detectable change; when the temperatures were above 60 degrees C, some fluorescence red-shift could be observed with a decrease in emission intensity. The inactivation rates (k(+0)) of free enzymes were faster than those of conformational changes during thermal denaturation at the same temperature. The rapid inactivation and slow conformational changes of phytase during thermal denaturation suggest that inactivation occurs before significant conformational changes of the enzyme, and the active site of this enzyme is situated in a relatively fragile region which makes the active site more flexible than the molecule as a whole.
Wang
Xiao-Yun
XY
College of Life Science, Shandong Agricultural University, Shandong Tai'an 271018, PR China.
Meng
Fan-Guo
FG
Zhou
Hai-Meng
HM
D010832:6-Phytase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D000135:Acid Phosphatase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D000814:Aniline Compounds / Q000737:chemistry; D000821:Animal Feed / Q000592:standards; D005503:Food Additives / Q000592:standards; D006358:Hot Temperature; D007700:Kinetics; D009943:Organophosphorus Compounds / Q000737:chemistry; D010758:Phosphorus / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence
D000814:Aniline Compounds; D005503:Food Additives; D009943:Organophosphorus Compounds; D010758:Phosphorus; C068798:4-aminophenylphosphate; D000135:Acid Phosphatase; D010832:6-Phytase