THE TECHNIQUES evolved and described in this work provide versatile and powerful tools for the investigation of enzymatic reactions. Off-line reaction monitoring using mass spectrometry allows a fast and almost completely automated determination of kinetic parameters. This has been shown for RNase T1 wild type, for which kinetic parameters for the transesterification of 5',3'-GpC to 2',3'-cGMP could be determined. Furthermore, kinetic parameters for the second reaction step, the hydrolysis of the cyclic 2',3' phosphodiester could be obtained, as well as parameters for the transesterification of 5',3'-ApC by the variant RNase T1 RV. The deviation of the parameters acquired with the parameters published so far may result from a difference in the reaction environment which has been chosen to improve the quality of the mass spectra. For future experiments, an physiological environment should be created to allow the comparison of the data obtained. However, the main field of application for this method is the automated screening of a large number of variants for altered specificities, thus applying an identical environent to all reactions under investigation. The method as presented in this work can be used for specificity screening without any alteration.
Due to the particular conditions under which the reactions had to be conducted, it has been impossible to use an internal standard for compound quantification. For this reason, a method has been evolved and applied in this work which uses the relative concentrations for quantification in off-line reaction monitoring.
On-line reaction monitoring using NANOES allows a closer look to the enzymatic reaction itself. This technique provides a continuous time-intensity curve for all compound involved in the reaction, products, intermediates, substrates and even modulators. The time-intensity data provides a deep insight into the mechanisms lying underneath the enzymatic reaction with very low sample amounts. Furthermore, the possibility to do tandem MS experiments with selected compounds during reaction monitoring is very useful for the elucidation of unknown reaction products and intermediates and therefore also for the elucidation of an unknown mechanism.
The NANOES source developed proved to be a reliable and versatile tool for mass spectrometric investigations; not only for on-line reaction monitoring but also for the investigation of biological compounds.
In contrast to the two techniques described above, CPA does
provide information on the specificity of the
enzyme regarding more native substrates. Using suitable substrates
with defined cleavage positions, it is possible to obtain information
on the middle- and long range specificity and on the reaction
mechanism of an enzyme. Performed with RNase T1, CPA revealed that RNase T1
does either have a preference for cleavage positions embedded into a
longer oligonucleotide chain or an yet unknown binding site beyond
. Further experiments using different substrates and also the
other techniques described above are necessary to obtain further
information.
The methods described in this thesis are tools for a further investigation of enzymatic reactions and can be used for both, the facilitated generation of new enzymes with altered features by new screening methods and the direct application of this techniques to the enzymatic reaction itself.