The Use of Isotope Effects to Determine Enzyme Mechanisms

Abstract

When our laboratory started to carry out kinetic experiments on enzyme-catalyzed reactions we focused originally on initial velocity studies in which the concentrations of substrates, products, and inhibitors were varied. The notation and theory for these types of experiments were published as three papers in Biochimica et Biophysica Acta that have received many citations over the years (1-3). We used these methods to study various enzymes over the next decade or so. Although we used isotopes to measure isotopic exchanges in creatine kinase (4), galactokinase (5), shikimate dehydrogenase (6), alcohol dehydrogenase (7), and isocitrate dehydrogenase (8) and to measure rates in NDP kinase (9), we did not determine isotope effects.However, in 1975 Dexter Northrop discovered how to exploit the Swain-Schaad relationship between deuterium and tritium isotope effects (10) to determine intrinsic isotope effects on the isotope-sensitive bond breaking step of an enzymatic reaction (11). The Swain-Schaad relationship says that the effect of tritium on a rate or equilibrium constant is the 1.442 power of the effect of deuterium substitution (this is derived from the relative masses of deuterium and tritium). Northrop assumed that there was no equilibrium isotope effect and thus that effects on V/K, the apparent first order rate constant at low substrate concentration and one of the independent kinetic constants, could be represented by Equation 1,

Inaccuracies in selected ion monitoring determination of isotope ratios obviated by profile acquisition: nucleotide 18O/16O measurements

“…The linearity of the data and the standard deviations of the replicates indicate that the precision of the isotope ratios obtained from the product ion should be sufficient to permit heavy atom isotope effects to be determined on nucleotides. Similar results were obtained for the protonated base product ion when [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] O]uridine was analyzed (data not shown). The procedures described to obtain and analyze IDs by tandem MS for nucleotides (and peptides [22]) will permit heavy atom isotope effect studies to be conducted on these biomacromolecular substrates.…”

“…18 O 1 ]pGp were purified by HPLC (300 mm × 3.9 mm, 10 μm C 18 packing) eluted isocratically using 50 mM diisopropylethylamine (Aldrich), 1% acetic acid in water (pH 4.3) as the mobile phase. For [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] O]uridine, samples were resolved by a gradient of acetonitrile in 0.2 M ammonium acetate (5% -20% acetonitrile in 60 min.). Appropriate fractions containing either uridine, UMP, TMP or pGp were collected.…”

“…The application of the analysis of isotope ratios by analysis of IDs was extended to tandemquadrupole time of flight mass spectrometry by analysis of [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] O]uridine and UM[ 18 O 1 ]P with an Applied Biosystems Q-STAR. All analyses were performed by direct infusion of the nucleoside/nucleotide in 50% acetonitrile 1% formic acid.…”

“…In the experimental cases we describe, each labeled compound contains a single enriched 18 O atom, so the prediction of the ID will be a sum of the unlabeled and labeled IDs, with the labeled ID weighted by the mol fraction ratio, X 18 / X 16 , as shown in equation 6. (6) To account for the mass offset introduced by the 18 O-label, n′ = n−2 (if n′ <0 then M n′ =0) while m 1′ and m 2′ are calculated with one less O atom. The experimentally sought value, X 18 / X 16 , is determined by minimizing the rms for the predicted and experimental IDs as shown in equation 7.…”

“…Heavy atom isotope effects generated by substituting 13 C, 15 N or 18 O are important tools for probing the mechanisms and transition states of chemical reactions [1], including enzymecatalyzed reactions [2][3][4][5][6][7]. Because the observed effects for isotopic substitution of heavy atoms are extremely small, typically a few percent, they cannot be determined by direct comparison of experimentally determined rate constants.…”