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| Time-dependent inhibition of 5alpha-steroid reductase |
| DynaFit tutorial |
Time-dependent inhibition of 5alpha-steroid reductase: A DynaFit tutorialBioKin Technical Note TN-2015-06Petr KuzmicBioKin Ltd. Draft - Rev. 1.01 - November 23, 2015 Download PDF
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AbstractExperimental data initially published by Moss, Kuzmic, et al. (Biochemistry 35, 3457, 1996) are re-analyzed here using a variety of techniques that were introduced into the DynaFit software (Anal. Biochem. 237, 260, 1996) since the original publications first appeared. The experimental system involves "slow, tight" inhibition of 5alpha-steroid reductase by a heterocyclic testosterone analog inhibitor. The results revealed a two-step kinetic mechanism of inhibition, involving essentially instantaneous equilibration to form initially a "loose" enzyme-inhibitor complex. The initial complex is characterized by equilibrium dissociation constant Kd(ini) = 5 nM. The initial complex rearranges over time to form the final "tight" complex, characterized by the total equilibrium dissociation constant Kd(tot) = 0.17 nM. The reversible rearrangement occurs within approximately four minutes. A detailed, step-by-step tutorial is presented, which shows (a) how to construct the appropriate DynaFit input file and (b) how best to interpret the numerical results.Keywordsenzyme kinetics; mathematics; DynaFit; tutorial; progress curve analysis; inhibition; slow-tight bindingHow to CiteKuzmic, P. (2015) Determination of substrate kinetic parameters from progress curve data, BioKin Technical Note TN-2015-06, BioKin Ltd., Watertown MA, [Online] www.biokin.com/TN/2015/06
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Sat Apr 20 18:22:31 2019