Off-rate kinetics

In far-from-equilibrium situations, the two binding partners are rapidly separated after unbinding and there is practically no recomplexation, that is, back-reaction. off(0) = off (/) relates the kinetic off-rate at a force f, kosif) to the off-rate at zero force, A off(0) by (6). 

In simple systems, the association rate constant, is the rate at which molecules collide in solution, and diffusion limits this value to 10 M -s L Note that the observed on-rate also depends on the ligand concentration it = ik [L], with units of s L The kinetic off-rate, is sensitive to the strength of the interaction in the ML complex the off-rate decreases as the strength of the interaction increases. Typical values for the off-rate vary from 10 to 10 s, corresponding to Kp values of lO to lO M. One of the strongest interactions that can be found in biological systems is the interaction between the protein avidin and the small molecule biotin the Kp for this interaction is 10M, corresponding to a phenomenally slow off-rate of 1 event every 115 days. 

HTS data and secondary screening for hit validation and determination of kinetic on/off rates. These data have been successfully incorporated into hit triage by enhancing the understanding of SAR differences between potential scaffolds [24]. In this example, those differences allowed informed decision making in the choice of which series to pursue and which to deprioritize. 

Using sub-ambient temperatures for preparing the protein-ligand equilibrium mixtures and for centrifugation of the GPC spin column, the dissociation rate constant decreases and the off-rate diminishes, thereby expanding the kinetic window observable with GPC spin column screening to even weaker binders with Kd values >20 pM. 

Deng and Sanyal have suggested that FAC is not applicable to ligands with low on- and off-rates [20]. As will be shown below, this is not true particularly when indirect FAC methods are applied, but it is also misleading in direct assays as described in this section. With flow-rate programming, for example, slow kinetics... 

The NO dissociation rate constants are summarized in Table HI (50) and are smaller than those seen with NO-metmyoglobin complexes. NP2 and NP3 (A ofr 0.1 s ) release NO approximately 10 times more slowly than NPl and NP4 kofs 2-3 s ) at pH 8.0, and the NO release rate for all nitrophorins decreases as the pH is lowered to 5.0. The NO release curves cannot be fit with a single exponential, indicating two off rates at each pH, as previously noted for both recombinant and insect-derived NPl (46), and which has also been recently reported for recombinant NP2 (145). The biphasic kinetics suggest the presence of slowly interconverting conformations. The values obtained are pH and protein dependent, ranging from 2.6 to 0.05 (Table HI) (50), values that are considerably slower than found for sperm whale metmyoglobin... 

One frequently encounters the case where the equilibrium dissociation constant (iQ, see above) is defined by microconstants with Tast rates on and off the receptor. However, any change in potency in a chemical series (affinity) must represent an increase in the on (k+i) rate or a decrease in the off rate (fe i). Occasionally, either by accident or design, the off rate is altered dramatically enough to redefine the receptor kinetics of the compound such that the rates influence the actual pharmacodynam-... 

The rate equation to be used for kinetic analysis of enzyme depletion is that for simple noncompetitive inhibition. If the Henderson equation or similar types are not employed, keep in mind that the inhibitor concentration [I] is the free inhibitor concentration. Determination of Ki may not be feasible if the rate assay is insensitive and requires an enzyme concentration much greater than K[. Alternatively, Ki may be obtained by measuring the on-off rate constants of the E l complex, provided the rate constants for any conformation change steps involved are also known. 

D.S. Ross et al, Study of the Basic Kinetics of Decomposition. . , AFRPL-70-29, SRI, Menlo Park, Contract F04611-69-C-0096 (1970) [From their work the authors conclude that there is no way to distinguish between the very low pressure pyrolysis reactions UDMH - NH3+CH2 N-CH2 (1) and UDMH ->(CH3)2N. +.NH2 (2). The reported pyrolysis fall-off rate constants kx are listed as log k(1 = 13.0 —... 

Figure 5. Rate constants for reactions of CO with mesoheme dimethyl ester and the indicated imidazoles in 2% aqueous CTAB at pH 7.3. On-rates and in M-1 sec f off-rates in sec 1. Data is from Ref. 20. Similar steric effects on kinetic constants were observed in benzene.

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