EVIDENCE SUPPORTING A ROLE FOR HISTIDINE-235 IN CATION-BINDING TO HUMAN 3-HYDROXY-3-METHYLGLUTARYL-COA LYASE

Histidine-235 of human 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase is the second basic residue in a conserved HXH motif, This residue is solvent accessible, readily reacting with the group specific reagent d iethyl pyrocarbonate. Site-directed mutagenesis has been employed to s ubstitute alanine or aspartate for H235, Characterization of the isola ted H235A and H235D lyase mutants indicates that their tertiary struct ure is substantially intact. The mutant proteins, like the wild-type e nzyme, are stoichiometrically modified by the affinity label, 2-butyno yl-CoA. Catalytic activity of the mutants is diminished by 15-fold and K-m for KMG-CoA elevated approximate to 4-fold in comparison with the values for wildtype enzyme. The function of H235 is suggested by inve stigation of the interaction of these enzymes with the dissociable div alent cation (e.g. Mg2+ or Mn2+) that is required for activity. ESR ex periments show that wild-type enzyme forms a stable binary EM complex . In contrast, H235A and H235D proteins do not efficiently form a bina ry complex. Significant interaction with cation (Mn2+) only occurs in the presence of the substrate analog, 3-hydroxyglutaryl-CoA. Similarly , when cation interaction is estimated in the presence of substrate us ing steady-state kinetic approaches, activator constants (K,) and diva lent cation K-m values are measurable but are elevated by 15-90-fold o ver comparable estimates for the wildtype enzyme. The data confirm our earlier suggestion that both protein and substrate contribute ligands to HMG-CoA lyase's divalent cation activator. More specifically, the current observations suggest that H235 has an important function in ca tion binding.