Effect of inhibitor binding to beta subunits of F1ATPase on enzyme thermostability: a kinetic and FT-IR spectroscopic analysis.
FT-IR analysis shows that treatment of F1ATPase with the inhibitors DCCD and Nbf-Cl, in the presence of saturating concentrations of ADP and AMP-PNP and in the absence of Mg2+, does not modify the secondary structure of the enzyme, but significantly modifies its compactness and thermal stability, although to different extents. Nbf-Cl causes a significant increase in stabilisation, in addition to that induced by nucleotides, while DCCD is less effective in this regard. Determination by HPLC of the exchange rate, in the absence of Mg2+, of tightly bound nucleotides of F1ATPase treated with the two inhibitors shows that DCCD does not significantly affect the exchange rate of ADP with AMP-PNP and vice versa in catalytic and non-catalytic tight sites, while Nbf-Cl selectively reduces the enzyme's capacity to exchange ADP bound in the tight catalytic site. It is suggested that the effects of DCCD, unlike those of Nbf-Cl, are closely related to the presence or absence of Mg2+.
Lippe
G
G
Dipartimento di Scienze e Tecnologie Biomediche, Università di Udine, Italy.
Tanfani
F
F
Di Pancrazio
F
F
Contessi
S
S
Bertoli
E
E
Dabbeni-Sala
F
F
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D006868:Hydrolysis; D007700:Kinetics; D008929:Mitochondria, Heart / Q000187:drug effects / Q000201:enzymology; D011485:Protein Binding / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
D004791:Enzyme Inhibitors; D000266:Adenylyl Imidodiphosphate; D004024:Dicyclohexylcarbodiimide; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan