Analysis of the pH-dependencies of the association and dissociation kinetics of HIV-1 protease inhibitors.
The kinetic constants for the interactions between HIV-1 protease and a selection of inhibitors were determined at different pH-values using a biosensor based interaction assay. Since this technique does not involve a substrate, it was possible to determine the pH-dependencies of the association and dissociation rates of an inhibitor, without the complication of a pH-dependent enzyme-substrate/product equilibrium. The importance of these interactions was evaluated by correlating the free energy changes upon association and dissociation of inhibitors with the predicted change in electrostatic properties of the interacting groups as a result of altered pH. It was found that the kinetic parameters varied with pH in a unique manner for all inhibitors, demonstrating that the kinetic features were associated with the specific structure of each inhibitor. Association and dissociation had different pH-profiles, indicating that the two processes proceeded by different pathways/mechanisms. The energy barrier for dissociation of the enzyme-indinavir complex increased with pH from 4.1 to 7.4, while it was generally reduced for the other inhibitors as the pH was increased from 5.1 to 7.4. The pH-dependent interactions involved in the recognition/binding of inhibitors and in the stabilization of the complex were identified by analysing three-dimensional structures of enzyme-inhibitor complexes. The interaction between the pyridine nitrogen of indinavir with Arg-8 was hypothesized to be responsible for the unique pH-dependency of indinavir. The analysis revealed features of interactions that are significant for understanding enzyme function and for optimization of new drug leads. It also highlighted the importance of environmental conditions on interactions.
Gossas
Thomas
T
Department of Biochemistry, Uppsala University, Uppsala, Sweden.
Danielson
U Helena
UH
D016333:HIV Protease / Q000737:chemistry / Q000378:metabolism*; D017320:HIV Protease Inhibitors / Q000737:chemistry / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D019469:Indinavir / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D013329:Structure-Activity Relationship
D017320:HIV Protease Inhibitors; D019469:Indinavir; D016333:HIV Protease