Control of Rabbit Liver Fructose-1, 6-diphosphatase Activity by Magnesium Ions

Abstract

EDTA at a concentration of 1μM produced a threshold effect in the activation of purified rabbit liver fructose-1, 6-diphosphatase [EC 3. 1. 3. 11] in the presence of 5m_M Mg²⁺ at pH 7.2. Without EDTA, biphasic activation curves were produced by Mg²⁺. A double-reciprocal plot of the data gave theK_m values corresponding to the two linear regions. They were 0.19 and 0.83m_M at pH 7.5, and 0.055 and 0.83m_M at pH 9.1.
In the presence of 5μ_M EDTA a sigmoidal curve was obtained for Mg²⁺ activation in the range of noninhibitory Mg²⁺ concentrations at pH 7.2. The apparentK_m value for Mg²⁺ was 0.15m_M, and the Hill coefficient was 2.0. At pH 9.1cooperativity among the Mg²⁺ sites disappeared, and the apparent K_m value for Mg²⁺ was 0.055m_M. These K_m values at pH 7.2 or 9.1 corresponded to the smaller of the biphasicK_m values obtained without EDTA.
In the absence of EDTA, no inhibition by Mg²⁺ was observed in the Mg²⁺ concentration range below 10m_M. In the presence of EDTA, the enzyme was inhibited markedly by Mg²⁺ at concentrations above 0.5m_M at pH 7.2, and was more sensitive to inhibition at pH 9.1. The effects of pH on the K_m value for Mg²⁺ activation and on the Mg²⁺ inhibitioncontributed to an apparent shift of the pH optimum for activity induced by EDTA.
Cooperative interaction amongfructose-1, 6-diphosphate sites was observed for the enzyme in the presence of EDTA. The Hill coefficient was approximately 1.8, and the apparent K_m value for the substrate was 0.74μ_M. EDTA appears to make liverfructose-1, 6-diphosphatase very sensitive to various effectors. It is suggested that Mg²⁺ serves as a regulatorfor the enzyme activity.