Unusually Large Deuterium Isotope Effects in Soybean Lipoxygenase Are Not Caused by a Magnetic Isotope Effect

Abstract

Herein, we report that soybean lipoxygenase (EC 1.13.1 1.12) exhibits a large deuterium kinetic isotope effect on the enzymecatalyzed oxygenation of the 1,Cdiene moiety in [11,1 l-2H2]linoleate.'V2 At 24.2 OC, we observe DVmax = 36 3 and D( Vmax/ K,) = 28 f 2. This is one of the largest kinetic isotope effects known for an enzymatic r e a~t i o n ,~ and it is difficult to explain simply by a difference in IH and 2H zero-point energies. In this report, we have eliminated the difference in nuclear magnetic moment between 1H and 2H as a cause of the large isotope effect.A previous report estimated DVmax = 8.7 0.4 and D(VmaJ K,) = 7.6 at 25 O C and pH 9.0, as determined by comparing the Supplementary Material Available: Isotopic data and calculations for determining the deuterium isotope effect by mass spectrometry; V , , and V, , , /K, , , vs magnetic field with arachidonate as substrate (2 pages). This material is contained in many libraries on microfiche, immediately follows this article in the microfilm version of the journal, and can be ordered from the ACS; see any current masthead page for ordering information. effects: = DVmax; V = D(Vmax/Km).

Kinetics of Manganese Lipoxygenase with a Catalytic Mononuclear Redox Center

“…The initial step of manganese lipoxygenase and iron lipoxygenase catalysis is removal of the pro-S hydrogen at C-11, which is accompanied by a large isotope effect (k H /k D Ͼ 15 for manganese lipoxygenase (6) and k H /k D ϭ 20 -80 for soybean lipoxygenase-1 (8,18,24)). The theoretically expected value of k H /k D is 7-10 (18).…”

Inter- and Intramolecular Experimental and Calculated Equilibrium Isotope Effects for (silox)2(tBu3SiND)TiR + RH (silox = tBu3SiO): Inferred Kinetic Isotope Effects for RH/D Addition to Transient (silox)2TiNSitBu3

“…Values of 27.4 (38) 41 and 28(2) (25(1)°C, pH ) 9) 42 have been independently obtained, and additional experiments suggest that a range of KIEs from 20 to 80 may characterize the oxidation under [LA]-dependent conditions, and that the KIEs increase when the temperature is raised. 41 Related experiments involving HPLC detection of LA oxidation by human 15-lipoxygenase (15-HLO) reveal a KIE of 47(7) at 38°C, which is comparable to the 48(5) measured for SLO.…”

Multidimensional Tunneling, Recrossing, and the Transmission Coefficient for Enzymatic Reactions

“…73,129,272 The pre-exponential isotope effect was found to be much greater (A H /A D = 27 after extrapolation to infinitely high T) than the quasiclassical limit, 78 and the activation energy is very small (ΔE act = 2.1 kcal/mol). 129 The mechanisms involving large magnetic isotope effects 273 or branch reactions 274 have been ruled out. Interestingly, the secondary KIE for the SLO reaction seems to be normal.…”