Comparison of the activity and conformation changes of lactate dehydrogenase H4 during denaturation by guanidinium chloride.
The inactivation and unfolding of lactate dehydrogenase (LDH) during denaturation by guanidinium chloride (GuHCl) under diverse conditions have been compared. Unfolding of the native conformation, as monitored by fluorescence and c.d. measurements, occurs in two stages with increasing GuHCl concentrations, and the inactivation approximately coincides with, but slightly precedes, the first stage of unfolding. The enzyme is inhibited to about 60-70% of its original activity by cross-linking with glutaraldehyde or in the presence of 1 M-(NH4)2SO4, with its conformation stabilized as shown by the requirement for higher GuHCl concentrations to bring about both inactivation and unfolding. Low concentrations of GuHCl (0.2-0.4 M) activate the cross-linked and the (NH4)2SO4-inhibited enzyme back to the level of the native enzyme. For the enzyme stabilized by (NH4)2SO4 or by cross-linking with glutaraldehyde, inactivation occurs at a markedly lower GuHCl concentration than that required to bring about its first stage of unfolding. It is concluded that the active site of LDH is situated in a limited region relatively fragile in conformation as compared with the molecule as a whole. The GuHCl activation of LDH stabilized in (NH4)2SO4 or by cross-linking with glutaraldehyde suggests that this fragility and consequently flexibility of the active site is required for its catalytic activity.
Ma
Y Z
YZ
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, China.
Tsou
C L
CL
D000818:Animals; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D007527:Isoenzymes; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D013552:Swine
D006146:Guanidines; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase; D019791:Guanidine