Triple Isotopic Labeling and Kinetic Isotope Effects: Exposing H-Transfer Steps in Enzymatic Systems

Abstract

Kinetic isotope effect (KIE) studies can provide insight into the mechanism and kinetics of specific chemical steps in complex catalytic cascades. Recent results from hydrogen KIE measurements have examined correlations between enzyme dynamics and catalytic function, leading to a surge of studies in this area. Unfortunately, most enzymatic H-transfer reactions are not rate-limiting and the observed KIEs do not reliably reflect the intrinsic KIEs on the chemical step of interest. Given their importance to understanding the chemical step under study, accurate determination of the intrinsic KIE from the observed data is essential. In 1975, Northrop developed an elegant method to assess intrinsic KIEs from their observed values [Northrop, D. B. (1975) Steady-state analysis of kinetic isotope effects in enzymic reactions, Biochemistry, 14, 2644–2651]. The Northrop method involves KIE measurements using all three hydrogen isotopes, where one of them serves as the reference isotope. This method has been successfully used with different combinations of observed KIEs over the years but criteria for a rational choice of reference isotope have never before been experimentally determined. Here we compare different reference isotopes (and hence distinct experimental designs) using the reduction of dihydrofolate and dihydrobiopterin by two dissimilar enzymes as model reactions. A number of isotopic labeling patterns have been applied to facilitate the comparative study of reference isotopes. The results demonstrate the versatility of the Northrop method, and that such experiments are limited only by synthetic techniques, availability of starting materials, and the experimental error associated with the use of distinct combinations of isotopologues.

The Effect of Protein Mass Modulation on Human Dihydrofolate Reductase

“…[Carbonyl- 14 C]-NADPH, 4R-[carbonyl- 14 C, 4- 2 H]-NADPH, 4R- 3 H-NADPH and DHF were synthesized and stored as per previously published protocols. 32–38 …”

“…Reactions were quenched at different time points by the addition of methotrexate and the depletion of tritium in the product was monitored as a function of fraction conversion in order to calculate the observed KIE on the second order rate constant k cat /K M . The observed H/T and D/T KIEs were used to calculate the intrinsic KIEs using a numerical solution to the modified Northrop equation (Eq 1): 38 $$\frac{{}^{\mathrm{T}}{\left(\text{V/K}\right)}_{\text{Hobs}}^{-1}-1}{{}^{\mathrm{T}}{\left(\text{V/K}\right)}_{\text{Dobs}}^{-1}-1}=\frac{{\left(k_{\mathrm{normalH}}/k_{\mathrm{normalT}}\right)}^{-1}-1}{{\left(k_{\mathrm{normalH}}/k_{\mathrm{normalT}}\right)}^{-1/33}-1}$$ where T (V/K) Hobs and T (V/K) Dobs are the observed H/T and D/T KIEs (also known as T ( k cat / K M ) obs ), respectively, and k H / k T is the intrinsic H/T KIE. The isotope effect on the activation parameters for the intrinsic KIEs was calculated by a non-linear fit of the data to the Arrhenius equation for intrinsic KIEs (Eq 2): $$\text{KIE}=\frac{k_{\mathrm{normall}}}{k_{\mathrm{normalh}}}=\frac{A_{\mathrm{normall}}}{{\mathrm{normalA}}_{\mathrm{normalh}}}\exp \left(\frac{-\mathrm{\Delta }{\mathrm{normalE}}_{\mathrm{normala}}}{\mathrm{normalRT}}\right)$$ where k l and k h are the rate constants for light and heavy isotopes, respectively, A l /A h is the isotope effect on the Arrhenius pre-exponential factor, Δ E a is the difference in energy of activation between the two isotopologues, R is the gas constant and T is the absolute temperature.…”

Atom Tunneling in Chemistry

“…[112] They all have their different kinetics and most of them are isotope-insensitive.T hus,t he apparent KIE based on either k cat or the catalytic efficiency k cat /K m differs from the intrinsic KIE of the isotope-sensitive step.T he intrinsic KIE can be estimated by using commitments to forward and reverse reactions. [8,10,24,113,114] Careful comparison between theory and experiment results in excellent agreements.F or example in the reaction with dihydrofolate reductase (DHFR), experimental intrinsic H/D KIEs [115,116] and values calculated at ah igh level of theory [117,118] both result in aK IE of 3.5 AE 0.1, independent of the temperature and almost independent of the pH value. [119] Apparent KIEs,m easured with different techniques result in KIEs less than 3.0, [120] which strongly indicates that steps other than the chemical transformation mask the KIE.…”

Preservation of Protein Dynamics in Dihydrofolate Reductase Evolution

“…Kinetic Isotope Effect Measurements-KIEs were measured as described for the WT and several mutants of ecDHFR (13,14,43 was co-purified using reverse phase HPLC, divided into aliquots containing at least 300,000 dpm of 14 C, and frozen in liquid nitrogen for short term storage at Ϫ80°C. KIE measurements were carried out in MTEN buffer (50 mM MES, 25 mM Tris, 25 mM ethanolamnine, and 100 mM NaCl) at pH 9.0 between 5-45°C.…”

“…Determination of the Intrinsic KIEs-Intrinsic KIEs were calculated as described previously (13,14,43), using a numerical solution of the modified Northrop equation (43,45), …”