Investigation of the oligomeric status of the peroxisomal isoform of human 3-hydroxy-3-methylglutaryl-CoA lyase.
Unprocessed 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase, retaining the mitochondrial signal sequence, has been proposed to correspond to a peroxisomal isoform. Using a modified expression plasmid and purification protocol, it is now possible to isolate substantial amounts (>10mg) of highly purified peroxisomal HMG-CoA lyase. These improvements facilitate more detailed protein chemistry approaches for characterization of the enzyme, which exhibits substantial (eightfold) dithiothreitol (DTT) stimulation of activity. The C323S mutant shows little DTT activation. Superose gel filtration chromatography data have prompted other investigators to hypothesize that the peroxisomal isoform is a monomer. This study confirms the elution properties presented in that earlier report, but also demonstrates anomalous elution up on Superose chromatography. Elution properties observed using a polyacrylamide resin (Bio-Gel P100) suggest a dimeric, rather than monomeric, enzyme. This observation has been further tested by protein chemistry experiments. The peroxisomal enzyme forms a covalently linked dimeric species upon crosslinking with dibromopropanone or o-phenylenedimaleimide or upon disulfide formation as a result of incubation with diamide. Cysteine-323 is required for intersubunit covalent crosslinking. Crosslinking efficiency is not dependent on HMG-CoA lyase protein concentration nor is it influenced by the presence of varying concentrations of an unrelated protein, such as ovalbumin. Sedimentation equilibrium analyses do not indicate a monomeric form of either human mitochondrial or human peroxisomal HMG-CoA lyase; the results suggest that these proteins are predominantly dimers. The retention of the basic N-terminal mitochondrial signal sequence in the peroxisomal HMG-CoA lyase isoform may influence elution from Superose gel filtration media but does not alter the oligomeric status of the enzyme.
Tuinstra
Robbyn L
RL
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Burgner
John W
JW
Miziorko
Henry M
HM
D000096:Acetone / Q000031:analogs & derivatives* / Q000494:pharmacology; D001671:Biochemistry / Q000379:methods; D002850:Chromatography, Gel; D002853:Chromatography, Liquid / Q000379:methods; D003545:Cysteine / Q000235:genetics; D019281:Dimerization; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D009154:Mutation; D007652:Oxo-Acid-Lyases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D020675:Peroxisomes / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D007527:Isoenzymes; D008301:Maleimides; D011994:Recombinant Proteins; D000096:Acetone; C047048:1,3-dibromo-2-propanone; D007652:Oxo-Acid-Lyases; C021410:3-hydroxy-3-methylglutaryl-coenzyme A lyase; D003545:Cysteine; C023860:N,N'-2-phenylenedimaleimide