Molecular Mechanisms of Enzyme Activation by Monovalent Cations

Abstract

Regulation of enzymes through metal ion complexation is widespread in biology and underscores a physiological need for stability and high catalytic activity that likely predated proteins in the RNA world. In addition to divalent metals such as Ca²⁺, Mg²⁺, and Zn²⁺, monovalent cations often function as efficient and selective promoters of catalysis. Advances in structural biology unravel a rich repertoire of molecular mechanisms for enzyme activation by Na⁺ and K⁺ Strategies range from short-range effects mediated by direct participation in substrate binding, to more distributed effects that propagate long-range to catalytic residues. This review addresses general considerations and examples.

Keywords: enzyme kinetics; evolution; potassium; pyruvate kinase; sodium; structure-function; thrombin.

FIGURE 1.

M⁺ coordination in proteins. The graphs show M⁺ and water coordination with O atoms participating in interactions for each coordination number from 4 (blue), 5 (red), 6 (green), 7 (purple), to 8 (orange). All structures annotated as containing Na⁺ (4,838) or K⁺ (1,534) in the Research Collaboratory for Structural Bioinformatics (RCSB) as of May 2016 were analyzed. Interactions related by symmetry were included (<1% of all observations). A single ionic bond equals one observation. O atoms within ligating distance represent the vast majority (79,567), followed by N atoms (5,711), K⁺ (1,030), and S atoms (302), with the remaining observations coming from other atom types. Water was the most commonly observed ligating residue (23,304) followed by Asp (7,144), Thr (5,613), Glu (5,289), and Ser (4,497), with all remaining observations coming from other protein residues or ligands. A, Na⁺–O coordination. The average bond distance across all coordination numbers is 2.4 ± 0.2 Å when only distances between 2.0 and 2.7 Å are considered (31,675), and 2.6 ± 0.3 Å for the entire range (47,892). For coordination numbers 4 and 5, there is a secondary peak around 2.75 Å, suggesting possible misidentification of water molecules as Na⁺. B, K⁺–O coordination. The average bond distances for coordination numbers 6 and 7 are 2.8 ± 0.2 and 2.9 ± 0.3 Å, respectively, over the entire range (20,494). C, water–O coordination. The coordinations of all crystallographic waters in the data set used for Na⁺ and K⁺ analysis (1,067,258) were calculated for comparison. For coordination number 4, shoulders around the peak correspond approximately to distances of 2.7 and 3.2 Å. The 2.7 Å shoulder is consistent with the secondary Na⁺–O peaks observed for coordination numbers 4 and 5, suggesting that stronger peaks in electron density maps and longer bond distances may refer to water molecules.

FIGURE 2.

Type I activated enzymes. A, diol dehydratase (Protein Data Bank (PDB) ID 1DIO) has K⁺ (yellow sphere) coordinated by five ligands from the protein and acting as bait for the two hydroxyl O atoms of substrate propanediol. B, BCKD kinase (PDB ID 1GJV) shown with substrate, relevant residues, K⁺ (yellow sphere), and Mg²⁺ (green sphere) bound to the Oδ1 atom of a conserved Asn and to the triphosphate moiety of ATP. C, GroEL (PDB ID 1KP8) shown with substrate, relevant residues, K⁺ (yellow sphere) and Mg²⁺ (green sphere). Nucleophilic attack on the Pγ of ATP is mediated by Asp⁵².

FIGURE 3.

Type II activated enzymes. A, ribokinase (PDB ID 1GQT) shown with substrate, relevant residues, and Cs⁺ (yellow sphere) that plays a functional role analogous to K⁺. The bound M⁺ is sequestered from solvent and contact with substrate, the ATP analog phosphomethylphosphonic acid adenylate ester (ACP). B, dialkylglycine decarboxylase (PDB ID 1DKA) shown with substrate, relevant protein residues, and K⁺ (yellow sphere). When Na⁺ replaces K⁺ in the site, a structural rearrangement brings the Oγ of Ser⁸⁰ in conflict with the phenyl ring of Tyr³⁰¹ that adopts a new conformation incompatible with substrate binding. C, Trp synthase (PDB ID 1BKS) shown with relevant protein residues and Na⁺ (yellow sphere) that binds to the β subunit, away from substrate and PLP, but near the tunnel that shuttles the indole for complexation with l-Ser.

FIGURE 4.

Molecular mechanism of Na⁺ activation in thrombin. A, structural determinants of Na⁺ activation in thrombin (PDB ID 1SG8). Shown are the Na⁺ (yellow sphere) coordination shell with water (red spheres) and relevant protein residues. Na⁺ binding is detected by Asp¹⁸⁹ and Asp²²¹ and then channeled through the corridors Cys¹⁹¹-Asp¹⁹⁴ and Ser²¹⁴-Cys²²⁰ to the catalytic residues Asp¹⁰² and Ser¹⁹⁵. The rotamer of Ser¹⁹⁵ is the end point of the Na⁺ effect, as demonstrated by the properties of the S195T mutant in panel B. The spatial separation of key residues responsible for transduction of the Na⁺ effect (arrows) underscores the contribution of backbone dynamics and overall conformational changes. B, contribution to Na⁺ activation of thrombin from residues in the two corridors Cys¹⁹¹-Asp¹⁹⁴ and Ser²¹⁴-Cys²²⁰ connecting the Na⁺ site to the catalytic residues Asp¹⁰² and Ser¹⁹⁵ (see also panel A; thrombin has no residue 218). Three residues are of particular importance, as their mutation has no effect on Na⁺ affinity but abrogates Na⁺ activation (81–83): Asp²²¹ supports one of the waters in the coordination shell, Asn¹⁴³ stabilizes the functional conformation of the backbone N atom of Gly¹⁹³ in the oxyanion hole, and Ser¹⁹⁵ is a member of the catalytic triad.