Substitution of His59 Converts CD39 Apyrase into an ADPase in a Quaternary Structure Dependent Manner

Abstract

The two transmembrane domains of CD39 ecto-apyrase regulate the formation of fully active homotetramers. We show that mutations in apyrase conserved region 1 (ACR1) have two dramatically different sets of effects determined by whether they occur in intact tetramers or in disrupted tetramers or monomers. In intact tetramers, substitution of H59 in the rat brain CD39 ACR1 with G or S abolishes more than 90% of the ATPase activity but less than 50% of the ADPase activity, converting the enzyme into an ADPase with relative ADP:ATP hydrolysis rates of 6:1 or 8:1, respectively. In contrast, the same substitutions in tetramers lacking either transmembrane domain, in monomers lacking both transmembrane domains, or in detergent-solubilized full-length monomers have no effect on ATPase activity and increase ADPase activity approximately 2-fold, resulting in equal ATPase and ADPase activities. N61R substitution has a much smaller effect on the ADPase:ATPase ratio in both cases. While the data for truncated and monomeric constructs are consistent with the proposed role of ACR1 as the beta-phosphate binding domain by analogy with the actin/hsp70/hexokinase superfamily, the finding that H59 substitutions in full-length CD39 primarily diminish the ATP hydrolysis rate suggests that ACR1 may play a different role in intact tetramers. We propose that CD39 uses different ATPase and ADPase mechanisms in different quaternary structure contexts, and that H59 in ACR1 plays a central role specifically in ATP hydrolysis in intact tetramers.

Bilayer Mechanical Properties Regulate the Transmembrane Helix Mobility and Enzymatic State of CD39

“…Alternatively, the mode of substrate binding itself may change in the relaxed state. A change in binding mode would be consistent with the documented change in hydrolysis mechanism with respect to ACR1 (24). Since ACR1 and ACR5 are adjacent to TM1 and TM2, and since we observe that both helices are required for the substrate sensitivity of either TM1 or TM2, it might be of interest to compare the role of ACR5 in substrate binding and hydrolysis in the tense and relaxed states.…”

“…In the native state, the transmembrane helices move relative to each other but substrate can reduce such motions; in contrast, when the enzyme is converted to the relaxed state by altering membrane properties, substrate can no longer stabilize the transmembrane helices. Previous experiments have indicated that soluble CD39 has an even higher substrate affinity than native CD39 and that both have a Km well below the concentrations used here (17,24,25), suggesting that the loss of coupling more likely reflects a change in the relationship between binding site and transmembrane domain dynamics than a lack of binding. One potential explanation is that altering the membrane reduces the energy barriers among helix orientations such that thermal energy is sufficient to overcome the stabilizing influence of substrate binding.…”

“…Nucleotidase assays were carried out in a 200 μL solution containing 50 mM Tris-HCl pH 7.5, 1 mM EDTA, I mM HEDTA, 2 mM ATP or ADP, and MgCl 2 or CaCl 2 to give the indicated concentrations of free Mg 2+ or Ca 2+ as calculated and described in ref 24. In most cases MgCl 2 was used in order to be consistent with the fact that the cytoplasmic side of the membrane is not usually exposed to high Ca 2+ concentrations and thus to avoid any potential nonphysiological interference of Ca 2+ with amphiphile insertion or distribution.…”

“…All of these enzymatic features line up one way when CD39 is in its native state and a different but consistent way regardless of whether the native state is disrupted by removing the N-terminal, the C-terminal, or both transmembrane helices or by detergent solubilization. Thus, native CD39 function requires both transmembrane helices to be present and in the membrane, and the state of the transmembrane helices governs which of at least two distinct functional states the active site resides in (24).…”

“…Energetically, altering membrane physical properties may alter the bilayer deformation energy for a given protein conformational change (29); if the energy difference between protein conformations is relatively low compared to the associated bilayer deformation energy, appropriate alteration of membrane properties can be sufficient to allow the conformational change. For CD39, the extracellular domain appears not to pose a barrier to whatever conformational change alters its function, since it achieves the altered state by default in the absence of transmembrane domains (24). The mobility discussed above suggests that the transmembrane helices themselves are also not optimized for strong association (28), potentially leaving the membrane as a significant factor keeping the protein in its native state.…”