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Slow Transients in the Activity of the NAD Malic Enzyme from Crassula¹

Department of Biochemistry, University of California, Riverside, California 92521

The NAD malic enzyme from Crassula argentea shows a slow reaction transient in the form of a lag before reaching a steady-state rate in assays. This lag, which has a half-time or ranging from seconds to many minutes under various conditions, poses problems in the interpretation of kinetic data with this enzyme. The NAD malic enzyme from Kalanchoë daigremontiana has a similar lag.

The lag is greatest in freshly prepared enzyme and diminishes with storage at –70°C, but the activity of the enzyme also diminishes with storage.

The lag is inversely proportional to the concentration of enzyme, both in the assay and in storage prior to assay. The lag is also inversely proportional to the concentration of malate used in the assay, which poses particular problems because the lag with low malate concentrations may be so long that activity begins to be lost before the steady-state rate is reached.

Various buffer ions produce different lags, but the lag with all buffers is longer than in the absence of buffer. The effectors CoA and SO₄^2– in the assay substantially decrease the lag. The lag is shorter with Mn²⁺ as the required divalent cation than when Mg²⁺ is used.

The response of enzyme activity to pH shows that the intrinsic activity is greater with magnesium than with manganese, although the rate actually attained is lower with Mg²⁺ because the pK values for the response to pH are closer together when that cation is used. The enzyme has a higher optimum pH and a broader response to pH when Mn²⁺ is used. The change in lag with pH follows the general pattern of activity with longer lags at intermediate pH values.

Preincubation of the enzyme with various reaction components and effectors reduces the lag, with NADH being the most effective. The presence of NADH in the assay is much more effective, but none of the treatments tried will completely eliminate the lag of freshly prepared enzyme.