Investigation of Conserved Acidic Residues in 3-Hydroxy-3-methylglutaryl-CoA Lyase: IMPLICATIONS FOR HUMAN DISEASE AND FOR FUNCTIONAL ROLES IN A FAMILY OF RELATED PROTEINS (original) (raw)

2003, Journal of Biological Chemistry

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase catalyzes the divalent cation-dependent cleavage of HMG-CoA to form acetyl-CoA and acetoacetate. In metaldependent aldol and Claisen reactions, acidic residues often function either as cation ligands or as participants in general acid/base catalysis. Site-directed mutagenesis was used to produce conservative substitutions for the conserved acidic residues Glu-37, Asp-42, Glu-72, Asp-204, Glu-279, and Asp-280. HMG-CoA lyase deficiency results from a human mutation that substitutes lysine for glutamate 279. The E279K mutation has also been engineered; expression in Escherichia coli produces an unstable protein. Substitution of alanine for glutamate 279 produces a protein that is sufficiently stable for isolation and retains substantial catalytic activity. However, thermal inactivation experiments demonstrate that E279A is much less stable than wild-type enzyme. HMG-CoA lyase deficiency also results from mutations at aspartate 42. Substitutions that eliminate a carboxyl group at residue 42 perturb cation binding and substantially lower catalytic efficiency (10 4-10 5-fold decreases in specific activity for D42A, D42G, or D42H versus wildtype). Substitutions of alanine for the other conserved acidic residues indicate the importance of glutamate 72. E72A exhibits a 200-fold decrease in k cat and >10 3-fold decrease in k cat /K m. E72A is also characterized by inflation in the K m for activator cation (26-fold for Mg 2؉ ; >200-fold for Mn 2؉). Similar, but less pronounced, effects are measured for the D204A mutant. E72A and D204A mutant proteins both bind stoichiometric amounts of Mn 2؉ , but D204A exhibits only a 2-fold inflation in K D for Mn 2؉ , whereas E72A exhibits a 12-fold inflation in K D (23 M) in comparison with wild-type enzyme (K D ‫؍‬ 1.9 M). Acidic residues corresponding to HMG-CoA lyase Asp-42 and Glu-72 are conserved in the HMG-CoA lyase protein family, which includes proteins that utilize acetyl-CoA in aldol condensations. These related reactions may require an activator cation that binds to the corresponding acidic residues in this protein family.

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