Enzymology kinetic experiments

A detailed model for the enzyme mechanism has been developed by Beinert and Kennedy and their collaborators on the basis of extensive spectroscopic studies, isotope labeling, kinetic experiments, several crystal structures, and site-directed mutagenesis experiments. As usual for an enzymological work in progress, the hardness of individual aspects of the model ranges from well established to conjectural. 

Capillary-in-capillary mixers were used for electrospray ionization mass spectrometry (ESI-MS), which allows one to perform on-line kinetic studies for a wide range of applications in chemistry, bioorganic chemistry, isotope exchange experiments and enzymology, just to name a few [133], ESI-MS is a method alternative to the traditionally employed quench-flow techniques with off-line analysis. 

The second method relies on the experimental determination of the kinetic parameters using techniques from biophysics or enzymology. Also in this case problems exist (1) the kinetic parameters are often determined under conditions different from the conditions in the cytoplasm (2) an enormous number of experiments need to be done, even for a network of moderate size, to determine all kinetic parameters experimentally. When the second method is used to parameterize a kinetic model then the resulting model is considered a silicon cell model. A number of silicon cell models exist [25-27, 29, 75-77]. 

The purpose of the present experiments is to characterize kinetics parameters of p-nitrophenyl-/3-D-galactoside hydrolysis with / -galactosidase. Students will find out how to use absorption spectroscopy to study enzymatic properties of an enzyme (see also Murata et al. 2003). Before entering the lab, students should be able to explain the Beer-Lambert-Bouguer law and the basics of enzymology. 

We hope to apply this method in the near future for enzymatic active centres. Replacement of the polar solvent with a fluctuating macromolecular environment, such as an enzyme, is methodologically a straightforward step but requires a lot of coding efforts. Quantum treatment of the nuclear motion is essential for calculation of the kinetic isotope effects that are of vital importance for enzymology. Very recently the H/D kinetic isotope effect h/ d = 80 was simulated with what is in excellent agreement with the experiment [36]. 

Lajis, N.H., Khan, M.N. A partial model for serine proteases kinetic demonstration of rate acceleration by intramolecular general base catalysis an experiment for a biochemistry/enzymology lab. Pertanika 1991,14, 193-199. 

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