Evaluation of 3-hydroxy-3-methylglutaryl-coenzyme A lyase arginine-41 as a catalytic residue: use of acetyldithio-coenzyme A to monitor product enolization.
3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase catalyzes the divalent cation-dependent cleavage of HMG-CoA to produce acetyl-CoA and acetoacetate. Arginine-41 is an invariant residue in HMG-CoA lyases. Mutation of this residue (R41Q) correlates with human HMG-CoA lyase deficiency. To evaluate the functional importance of arginine-41, R41Q and R41M recombinant mutant human HMG-CoA lyase proteins have been constructed, expressed, and purified. These mutant proteins retain structural integrity based on Mn(2+) binding and affinity labeling stoichiometry. R41Q exhibits a 10(5)-fold decrease in V(max); R41M activity is >or=10-fold lower than the activity of R41Q. Acetyldithio-CoA, an analogue of the reaction product, acetyl-CoA, has been employed to test the function of arginine-41, as well as other residues (e.g., aspartate-42 and histidine-233) implicated in catalysis. Acetyldithio-CoA supports enzyme-catalyzed exchange of the methyl protons of the acetyl group with solvent; exchange is dependent on the presence of Mg(2+) and acetoacetate. In comparison with wild-type human enzyme, D42A and H233A mutant enzymes exhibit 4-fold and 10-fold decreases, respectively, in the proton exchange rate. In contrast, R41Q and R41M mutants do not catalyze any substantial enzyme-dependent proton exchange. These results suggest a role for arginine-41 in deprotonation or enolization of acetyldithio-CoA and implicate this residue in the HMG-CoA cleavage reaction chemistry that leads to acetyl-CoA product formation. Assignment of arginine-41 as an active site residue is also supported by a homology model for HMG-CoA lyase based on the structure of 4-hydroxy-2-ketovalerate aldolase. This model suggests the proximity of arginine-41 to other amino acids (aspartate-42, glutamate-72, histidine-235) implicated as active site residues based on their function as ligands to the activator cation.
Tuinstra
Robbyn L
RL
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Wang
Chang-Zeng
CZ
Mitchell
Grant A
GA
Miziorko
Henry M
HM
D000105:Acetyl Coenzyme A / Q000737:chemistry; D000101:Acetyl-CoA C-Acetyltransferase / Q000737:chemistry; D000214:Acyl Coenzyme A / Q000737:chemistry*; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D041961:Deuterium Exchange Measurement; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D007652:Oxo-Acid-Lyases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011522:Protons
D000214:Acyl Coenzyme A; D011522:Protons; C059703:acetyldithio-coenzyme A; C008047:3-hydroxy-3-methylglutaryl-coenzyme A; D000105:Acetyl Coenzyme A; D001120:Arginine; D000101:Acetyl-CoA C-Acetyltransferase; D007652:Oxo-Acid-Lyases; C021410:3-hydroxy-3-methylglutaryl-coenzyme A lyase