Kinetic isotope effect (KIE) measurements are a powerful tool for studying enzyme mechanisms; they can provide insights into microscopic catalytic processes and even structural constraints for transition states. In this work, we introduce a novel NMR-based assay able to measure small 13C competitive KIEs (CKIEs) continuously with excellent precision using 1D 1H NMR. The KIE is acquired in a single competitive assay, which requires relatively small amounts of each substrate isotopologue. The approach was demonstrated with the solution reactions of S-adenosyl-methionine (AdoMet) in Chapter 3, and with two enzymes, human catechol-O-methyltransferase (COMT) in Chapter 4 and E. coli AdoMet synthetase in Chapter 5.AdoMet synthetase performs a key role in producing AdoMet using methionine and ATP. The secondary CKIE on this reaction was found to be inverse, but the observed CKIEs do not remain stable throughout the reaction progress. These results suggest that the reaction is kinetically complex, and more information about the commitment of each step will be required to confirm our results.The rate constants and CKIEs on epimerisation and hydrolysis of (S,S)-AdoMet in solution were investigated. Rate constants were in good agreement with literature values, and the CKIEs were shown to be small and not significantly different to unity. This work demonstrated the general utility of the NMR assay to measure precise CKIEs, but also highlights potential problems with using 1H NMR as the error in the CKIEs values was dominated by uncertainty in the quantification of the diastereomers of the light isotopologue due to overlapping NMR resonances.COMT performs an important role in the metabolism of catecholamine neurotransmitters. Current COMT inhibitor drugs are not without problems, and new drug design requires a better understanding of the COMT-catalysed methyl transfer reaction mechanism. An NMR pulse sequence was developed to study the methyl transfer reaction catalysed by human COMT. Based on the J-coupling constant of the S- Adenosyl-L-homocysteine (a product) methylene protons, this pulse sequence is able to suppress 99% of AdoHomo methylene signals, while maintaining flat baseline and phased signals. We hypothesized that the magnitude of the 1o KIE would correlate with the rate of methyl transfer, and showed this is the case. Regiospecific CKIEs were measured on the production of meta and para O-methyldopamine products and the CKIEs = 1.075 ± 0.018 and 1.031 ± 0.007 for the slower and faster reactions, respectively. The temperature dependence of the CKIE was also measured for the firsttime and found to be modest with a difference in activation energy of 0.11 ± 0.1 kJ mol- 1.
- Kinetic Isotope Effects