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Estimating kinetic and thermodynamic parameters from single molecule enzyme-inhibitor interactions.

ABSTRACT: We report the application of recently developed microscopic models to estimate the apparent kinetic and thermodynamic parameters in a single molecule force spectroscopy study of the carbonic anhydrase enzyme and a complementary sulfonamide inhibitor. The most probable rupture force for the enzyme-inhibitor interaction shows a nonlinear dependency on the log-loading rate. Estimates for the kinetic and thermodynamic parameters were obtained by fitting the nonlinear dependency to linear cubic potential and cusp potential models and compared to the standard Bell-Evans model. The reliability of the estimated parameters was verified by modeling the experimental rupture force distributions by the theoretically predicted distributions at rupture. We also report that linkers that are attached to the enzyme and inhibitor show appreciable effects on the apparent kinetic and thermodynamic parameters.

SUBMITTER: Porter-Peden L

PROVIDER: S-EPMC2586077 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Estimating kinetic and thermodynamic parameters from single molecule enzyme-inhibitor interactions.

Porter-Peden Laura L Kamper Sarah G SG Wal Mark Vander MV Blankespoor Ronald R Sinniah Kumar K

Langmuir : the ACS journal of surfaces and colloids 20080923 20

We report the application of recently developed microscopic models to estimate the apparent kinetic and thermodynamic parameters in a single molecule force spectroscopy study of the carbonic anhydrase enzyme and a complementary sulfonamide inhibitor. The most probable rupture force for the enzyme-inhibitor interaction shows a nonlinear dependency on the log-loading rate. Estimates for the kinetic and thermodynamic parameters were obtained by fitting the nonlinear dependency to linear cubic poten ...[more]

PMID: 18808161

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Project description:Sponge-like biomaterials formed from silk fibroin are promising as degradable materials in clinical applications due to their controllable breakdown into simple amino acids or small peptides in vivo. Silk fibroin, isolated from Bombyx mori silkworm cocoons, can be used to form sponge-like materials with a variety of tunable parameters including the elastic modulus, porosity and pore size, and level of nanocrystalline domains. These parameters can be independently tuned during formulation resulting in a wide parameter space and set of final materials. Determining the mechanism and rate constants for biomaterial degradation of these tunable silk materials would allow scientists to evaluate and predict the biomaterial performance for the large array of tissue engineering applications and patient ailments a priori. We first measured in vitro degradation rates of silk sponges using common protein-degrading enzymes such as Proteinase K and Protease XIV. The concentration of the enzyme in solution was varied (1, 0.1, 0.01 U/mL) along with one silk sponge formulation parameter: the level of crystallinity within the sponge. Additionally, two experimental degradation methods were evaluated, termed continuous and discrete degradation methods. Silk concentration, polymer chain length and scaffold pore size were held constant during experimentation and kinetic parameter estimation. Experimentally, we observed that the enzyme itself, enzyme concentration within the bulk solution, and the sponge fabrication water annealing time were the major experimental parameters dictating silk sponge degradation in our experimental design. We fit the experimental data to two models, a Michaelis-Menten kinetic model and a modified first order kinetic model. Weighted, non-linear least squares analysis was used to determine the parameters from the data sets and Monte-Carlo simulations were utilized to obtain estimates of the error. We found that modified first order reaction kinetics fit the time-dependent degradation of lyophilized silk sponges and we obtained first order-like rate constants. These results represent the first investigations into determining kinetic parameters to predict lyophilized silk sponge degradation rates and can be a tool for future mathematical representations of silk biomaterial degradation.

Dataset Information

Estimating kinetic and thermodynamic parameters from single molecule enzyme-inhibitor interactions.

Publications

Estimating kinetic and thermodynamic parameters from single molecule enzyme-inhibitor interactions.

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