Identification of the site of acetyl-S-enzyme formation on avian liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase

Abstract

Avian liver mitochondrial hydroxymethylglutaryl-CoA synthase contains an active-site cysteine involved in forming the labile acetyl-S-enzyme intermediate. Identification of and assignment of function to this cysteine have been accomplished by use of an experimental strategy that relies upon generation and rapid purification of the S-acetylcysteine-containing active-site peptide under mildly acidic conditions that stabilize the thioester adduct. Automated Edman degradation techniques indicate the peptide's sequence to be Arg-Glu-Ser-Gly-Asn-Thr-Asp-Val-Glu-Gly-Ile-Asp-Thr-Thr-Asn-Ala-Cys-Tyr. The acetylated cysteine corresponds to position 129 in the sequence deduced from cDNA data for the hamster cytosolic enzyme [Gil, G., Goldstein, J.L., Slaughter, C.A., & Brown, M.S. (1986) J. Biol. Chem. 261, 3710-3716]. The acetyl-peptide sequence overlaps that reported for a tryptic peptide that contains a cysteine targeted by the affinity label 3-chloropropionyl-CoA [Miziorko, H. M., & Behnke, C. E. (1985) J. Biol. Chem. 260, 13513-13516]. Thus, availability of these structural data allows unambiguous assignment of the acetylation site on the protein as well as a refinement of the mechanism explaining the previously observed affinity labeling of the enzyme.

The Influence of Conserved Aromatic Residues in 3-Hydroxy-3-methylglutaryl-CoA Synthase

“…The stoichiometry of noncovalent acetoacetyl-CoA binding was determined using a modification of the procedure of Vollmer et al (18). The enzyme (150 g) in 100 mM sodium phosphate, pH 7.0, was incubated for 5 min at 30°C.…”

“…6000 transients were acquired for each spectrum. Samples contained 0.5 mM enzyme sites and 1.5 mM [1,2- For measurement of 18 O differential isotope shifts of the mutant or wild-type [1,2-…”

“…Spectra were recorded at 23°C. Samples were prepared by dissolving matched, buffered, freeze-dried proteins in either reagent grade 16 O water, supplemented with 10% D 2 O, or 18 Oenriched (95%) water supplemented with 10% D 2 O. Final samples (0.5 ml) contained 0.6 mM enzyme sites and 1.3 mM [1,2-…”

“…A minimum of two spectra were consecutively run for each 16 O-or 18 O-containing sample. Additional spectra for 18 O samples were run after overnight incubation (minimum 12 h) with no further changes in isotope shifts being detected.…”

“…A minimum of two spectra were consecutively run for each 16 O-or 18 O-containing sample. Additional spectra for 18 O samples were run after overnight incubation (minimum 12 h) with no further changes in isotope shifts being detected. Reported isotope shifts are not corrected for dilution of Targets-Alignments of 28 deduced sequences for HMG-CoA synthases indicate that six aromatic residues are invariant or highly conserved.…”