Oxy 32 oxygen monitoring system

To investigate the effects of metal introduction on the kinetic properties of E-Zn²⁺ and E-Co²⁺, the enzymes were analyzed under steady-state conditions by monitoring the initial rates of oxygen consumption with a computer-interfaced Oxy-32 oxygen-monitoring system (Hansatech Instruments Ltd) thermostated with a water bath. The steady-state kinetic parameters of E-Zn²⁺ or E-Co²⁺ were determined by varying the concentrations of the reducing substrate D2HG (0.025–0.4 mM) or d-malate (1.6–40 mM) and the artificial electron acceptor PMS (0.02–0.5 mM) in 25 mM NaPO₄, pH 7.4, and 25 °C with ∼7 nM enzyme.
Data analysis was conducted using the KaleidaGraph software (Synergy Software) and Enzfitter software (Biosoft). To determine the steady-state kinetic mechanism of E-Zn²⁺, the best fit of the initial rates of enzyme reaction was obtained using the equation that describes a ping–pong bi–bi steady-state process, consistent with the data obtained previously for PaD2HGDH purified without metals (18 (link)).

To investigate the effect of divalent cations on the activity of natively expressed PaD2HGDH purified without metals, the enzyme's activity was analyzed by adding exogenous amounts of 100 μM chloride salts of Co²⁺, Ca²⁺, Zn²⁺, Mn²⁺, Mg²⁺, Fe²⁺, Cd²⁺, Cu²⁺, or Ni²⁺ to an enzyme reaction mixture. The enzyme reaction mixture comprised of buffer, PaD2HGDH, reducing substrate, and an artificial electron acceptor. Activity assays for PaD2HGDH with 5 mM d-malate as a substrate and 1 mM PMS as the electron acceptor were measured by monitoring the initial oxygen consumption rates with a computer-interfaced Oxy-32 oxygen-monitoring system (Hansatech Instruments Ltd) after the exogenous addition of metals in 25 mM Tris–Cl, pH 7.4 and 25 °C. The data were analyzed using Microsoft Excel (data not shown).

To investigate the effect of Zn²⁺ incorporation on the O₂ reactivity of PaD2HGDH, the enzymatic activity of E-Zn²⁺ with molecular oxygen was measured by monitoring the initial oxygen consumption rate with a computer-interfaced Oxy-32 oxygen-monitoring system (Hansatech Instruments Ltd) at 25 °C. To test for oxygen reactivity, the reaction mixture contained 40 nM enzyme and d-malate between 1.6 and 40 mM in 25 mM NaPO₄, pH 7.4. After ∼3 min of reaction time, 1 mM PMS was added to the reaction mixture to regenerate the oxidized state of the enzyme at the expense of O₂. The instantaneous reoxidation of the enzymatically reduced PMS by molecular oxygen was then observed (data not shown).