Properties of Purified Malic Enzyme in Relation to Crassulacean Acid Metabolism

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Abstract
Malic enzyme from leaves of the Crassulacean species Bryophyllum tubiflorum was purified more than 1200‐fold by a rather simple procedure involving repeated fractionation with ammonium sulphate and gel‐filtration. A specific activity (international standards) of 68 was obtained. The molecular weight of the enzyme, measured by the gel‐filtration method, was 237000.Thiol activation is required for catalytic activity. The activity is completely dependent on divalent cations; greatest activity was obtained with Mn2+, an equimolar amount of Mg2+ was 86% as effective. Maximum activity was obtained at about pH 7.2. At 30 °C and pH 7.2 the Km for Mn2+ was 0.46 mM, that for NADP was 14 μM, and that for malate amounted to 0.37 mM. The Km for malate was temperature‐dependent: it remained constant in the temperature range from 17 to 39 °C, but below 17 °C and beyond 39 °C the affinity for malate decreased and a Km‐value of 0.94 mM was recorded. The enzyme was completely NADP‐specific and was stable at temperatures up to 55 °C; maximum activity was recorded at 50 °C.No effects of monovalent cations such as Na+, K+, Li+, or NH4+ could be recorded. The enzyme was inhibited by coenzyme A and thiamine pyrophosphate, but not by acetyl‐coenzyme A. The effects of temperature as well as the inhibition by coenzyme A and thiamine pyrophosphate fit the physiology of Crassulacean acid‐metabolism.