Comparison of inactivation and conformational changes of D-glyceraldehyde-3-phosphate dehydrogenase during thermal denaturation.
The inactivation of D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating) EC 1.2.1.12) (GAPDH) during thermal denaturation has been compared to its dissociation-aggregation measured by light scattering and changes in secondary structure measured by CD in the far ultraviolet. The inactivation at 38.5 degrees C consists of two stages. The rate of the first stage is too fast to be followed by conventional methods. The extent of this fast stage inactivation increases with increasing temperature and, more markedly, with increasing pH. At this stage, the inactivation is reversible and no appreciable dissociation or change in secondary structure can be detected. The secondary structure of the enzyme is relatively heat stable, showing no appreciable change at 38.5 degrees C. At this temperature, the enzyme first dissociates within several minutes probably into dimers and with prolonged heating, it becomes irreversibly aggregated. The above results are in accord with the earlier suggestion, based on results obtained during denaturation of a number of enzymes by guanidine hydrochloride (GdnHCl) and urea, that for some enzymes the active site is situated in a region more susceptible to perturbation than the molecule as a whole (Tsou, C.-L. (1986) Trends Biochem. Sci. 11, 427).
Lin
Y Z
YZ
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, China.
Liang
S J
SJ
Zhou
J M
JM
Tsou
C L
CL
Wu
P Q
PQ
Zhou
Z K
ZK
D000818:Animals; D001051:Apoenzymes; D002942:Circular Dichroism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D009132:Muscles / Q000201:enzymology; D008121:Nephropidae; D011487:Protein Conformation; D011489:Protein Denaturation; D012542:Scattering, Radiation; D013329:Structure-Activity Relationship
D001051:Apoenzymes; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases