title: Modeling of a mutation responsible for human 3-hydroxy-3-methylglutaryl-CoA lyase deficiency implicates histidine 233 as an active site residue.
abstract: 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase is inactivated by diethyl pyrocarbonate (DEPC); activity can be fully restored by incubation with hydroxylamine. Protection against DEPC inactivation is afforded by a substrate analogue, suggesting an active site location for a DEPC target. Included in the inherited defects that map within the HMG-CoA lyase gene is a point mutation that results in an arginine substitution for histidine 233, one of only two invariant histidines. These observations prompted a functional test of the importance of His-233. The mutant lyases H233R, H233A, and H233D were overexpressed in Escherichia coli, isolated, and kinetically characterized. In H233D, DEPC targets one less histidine than was measured using wild-type lyase, supporting the assignment of wild-type lyase His-233 as one of the DEPC targets. Substitution of His-233 results in diminution of activity by approximately 4 orders of magnitude. Km values of the mutant lyases for both substrate HMG-CoA and activator divalent cation (Mg2+ or Mn2+) are comparable to the values measured for wild-type enzyme, indicating that these enzymes retain substantial structural integrity. This conclusion is reinforced by the observation that the affinity label, 2-butynoyl-CoA, stoichiometrically modifies the mutant lyases, indicating that they contain a full complement of active sites. In view of these data suggesting that the structures of these mutant lyases closely approximate that of the wild-type enzyme, their observed 10(4)-fold diminution in catalytic efficiency supports assignment to His-233 of a role in the chemistry of HMG-CoA cleavage.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
authors.forename: J R
authors.initials: JR
authors.lastname: Roberts
authors.forename: G A
authors.initials: GA
authors.lastname: Mitchell
authors.forename: H M
authors.initials: HM
authors.lastname: Miziorko
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008661:Metabolism, Inborn Errors / Q000201:enzymology / Q000235:genetics*; D007652:Oxo-Acid-Lyases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation
chemical_list: D000214:Acyl Coenzyme A; D004791:Enzyme Inhibitors; C008047:3-hydroxy-3-methylglutaryl-coenzyme A; D006639:Histidine; D007652:Oxo-Acid-Lyases; C021410:3-hydroxy-3-methylglutaryl-coenzyme A lyase; D004047:Diethyl Pyrocarbonate