Jr. Roberts et Hm. Miziorko, EVIDENCE SUPPORTING A ROLE FOR HISTIDINE-235 IN CATION-BINDING TO HUMAN 3-HYDROXY-3-METHYLGLUTARYL-COA LYASE, Biochemistry, 36(24), 1997, pp. 7594-7600
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.