Analysis of the inhibitory non-catalytic ADP binding site on mitochondrial F1, using NAP3-2N3ADP as probe. Effects of the modification on ATPase and ITPase activity
| Autor: | Aloysius F. Hartog, C.M. Edel, Jan A. Berden |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
chemistry.chemical_classification
Non-catalytic site biology Photoaffinity labeling NAP3-2-azido-ADP Stereochemistry Cooperativity ATPase Biophysics Substrate (chemistry) Cell Biology Ligand (biochemistry) Biochemistry F1 chemistry Adenine nucleotide Hysteretic inhibition biology.protein ADP binding Nucleotide Tight binding |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1229:103-114 |
| ISSN: | 0005-2728 |
| DOI: | 10.1016/0005-2728(94)00194-a |
| Popis: | The ADP analogue NAP3-2N3ADP is able to bind to one or two high-affinity sites on mitochondrial F1-ATPase, depending on the nucleotide content of the F1 preparation. In both cases studied (enzyme with three bound nucleotides and enzyme with four bound nucleotides), the binding is accompanied by the exchange of one tightly-bound adenine nucleotide and nearly complete inhibition of the ATPase activity upon UV illumination. In both cases the ADP-analogue binds at a high-affinity catalytic site, replacing a bound nucleotide. The apparent KD value for the exchange equals 25–30 μM, but the newly bound ligand does not dissociate. With F1 containing 3 bound nucleotides NAP3-2N3ADP is able to bind to a second high-affinity site as well. This binding induces already in the absence of illumination 45% inhibition of the ATPase activity. The additionally bound molecule does not exchange within a short period of turnover with Mg-ATP. Therefore it has to be bound at a slowly exchangeable non-catalytic site, with a regulatory influence on the activity of the enzyme. Binding of NAP3-2N3ADP to this non-catalytic site is influenced by the presence of Mg2+ or EDTA: tight binding requires Mg2+ and in the absence of Mg2+ and presence of EDTA the ligand is removed from this site relatively easily, just like ADP. The presence of EDTA instead of Mg2+ lowers the measured affinity of this site for NAP3-2N3ADP with a factor 5. Kinetic measurements after an incubation of F1 with NAP3-2N3ADP show a decrease of the Vmax with ATP as substrate, without effect on the two measured Km values. With ITP as substrate, however, incubation of F1 with NAP3-2N3ADP results in an increase of the Km values, without effect on the Vmax. Comparison of our data with the literature shows that this non-catalytic site is not the site responsible for hysteretic inhibition by ADP. We conclude that this latter form of inhibition is observed when ADP or a suitable analogue is bound at the first (potentially catalytic) β-site, in disagreement with the conclusions of Jault and Allison (J. Biol. Chem. 269 (1994) 319–325). |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect