Acidic phospholipids may inhibit rat brain hexokinase by interaction at the nucleotide binding site

Rat brain hexokinase (ATP:D-hexose 6-phosphotransferase; EC 2.7.1.1) is inhibited by acidic phospholipids such as phosphatidylinositol, phosphatidylserine, and cardiolipin. Several aspects of this inhibition are atypical when compared to inhibition by established reversible inhibitors of this enzyme such as the product, Glc-6-P. Maximal inhibition is attained rather slowly (approximately 30 min at 22 degrees C), and is not reversed by simple dilution of the enzyme-lipid mixture. Ligands such as ATP or Glc-6-P can protect the enzyme against inhibition by acidic phospholipids; addition of protective ligands after mixing of enzyme and lipids does not, however, reverse inhibition that occurred prior to ligand addition. Inhibition can be prevented but not reversed by elevated (0.1-0.2 M) [NaCl], indicating a probable role for electrostatic forces in the interaction of lipid with enzyme. Greater inhibition is seen at 22 degrees C than at 3-4 degrees C, suggesting that hydrophobic interactions may also be involved. It is suggested that acidic phospholipids inhibit brain hexokinase by binding at the nucleotide-binding site of the enzyme. The effectiveness of ATP (or the ATP analog, Cibacron Blue) in protecting against inhibition by acidic phospholipids is attributed to direct competition between ATP and the phospholipid for a common binding site. The effectiveness of Glc-6-P (or analogs) in preventing the inhibition is attributed to a conformational change, induced by the binding of this ligand, which prevents binding of ATP or acidic phospholipids to the enzyme. The pH dependency of the inhibition has suggested involvement of the protonated form of a dissociable group (pK approximately 7) on the enzyme in the interaction with acidic phospholipids; this may be the histidyl residue implicated by Solheim and Fromm [Biochemistry 19, 6074-6080 (1984)] in the binding of ATP to brain hexokinase. Structural similarities in the nucleotide-binding sites of several nucleotide-binding enzymes suggest that similar inhibition by acidic phospholipids may be seen with other enzymes of this type; there are already some reports to this effect.