Measurement of 2H distribution in natural products by quantitative 2H NMR: An approach to understanding metabolism and enzyme mechanism?

Abstract

During the biosynthesis of natural products, the intra-molecular distribution of isotopes is introduced as a result of different isotope effects associated with the reactions involved. Due to the sensitivity of certain enzymes to the presence of a heavy isotope, the isotope selection effects related to some transformations can be high, especially for hydrogen. The effect of a series of isotope effects specific to each enzyme-catalysed step are additive during a biosynthetic pathway, leading to fractionation of the isotopes between the starting substrate and the final product. As the individual reactions are acting on different positions in the substrate, the net effect is a non-statistical distribution of isotope within the final product. Quantitative 2H NMR spectroscopy can be used to measure the distribution of 2H at natural abundance in natural products. In the first example, the fermentation of glucose is examined. Glucose can act as a primary carbon source for a wide range of fermentation products, produced by a variety of pathways. In many cases, competing pathways are active simultaneously. The relative fluxes are influenced by both environmental and genetic parameters. Quantitative 2H NMR spectroscopy is being used to obtain mechanistic and regulatory information about isotopic fractionation from glucose during such fermentations. Quantitative 2H NMR spectroscopy can also be used to examine the fractionation in 2H that occurs in long-chain fatty acids during chain elongation and oxygenation. It has been found that the (2H/1H) ratio shows an alternating pattern along the length of the chain and that the residual hydrogen atoms at the sites of desaturation are asymmetrically impoverished. The extent to which the non-statistical distribution of isotopes can be related to the mechanism of enzymes involved in the biosynthetic pathway via kinetic isotopic effects will be discussed.