Purification and crystallization of thiolase; study of its action mechanism

Abstract

Thiolase was purified 600–700 fold from a crude extract of pig heart by use of differential adsorption on Al(OH)3 gel, ammonium sulfate fractionation and ion exchange chromatography on DEAE‐Sephadex, CM‐Sephadex and phosphorylated cellulose. The enzyme preparation is homogeneous by the following criteria: sedimentation analysis and electrophoresis on polyacryl‐amide gel and on cellulose acetate membranes. The isoelectric point of the protein was found to be approximately pH 7.2–7.3.The enzyme crystallized from ammonium sulfate solutions as thin plates of irregular shape. Crystalline thiolase stored for 18 months at —10° did not show any loss of activity.The thiolytic activity and the acyl transferase activity of the enzyme could be demonstrated separately.The thiolytic activity was demonstrated by the specific incorporation of 14C from [1‐14C]‐acetyl‐CoA into the carboxyl position of unlabelled acetoacetyl‐CoA on incubation with the enzyme.The acyl transferase activity was studied using either [1‐14C]acetyl‐CoA and pantethein as substrates or 14C‐labelled CoA and acyl‐CoA derivatives of various chain length. A remarkable specificity was found for the transfer of the acetyl group.Thiolase activity and acyl transferase activity of the enzyme were diminished with the same kinetics during heat treatment or incubation with N‐ethylmaleimide, p‐chloromercuribenzoate or iodoacetamide. This indicates that both activities belong to the same protein. Inhibition of thiolase and acyltransferase activities by iodoacetamide (5 × 10−5 M) could be prevented by preincubation of the enzyme with acetoacetyl‐CoA or acetyl‐CoA.The acyl transfer is the rate determining step when the thiolase reaction operates in the direction of chain cleavage.Incubation of thiolase with acetyl‐CoA results in binding of acetyl groups to the protein. The uptake of acetyl groups is dependent upon the specific activity of the enzyme and corresponds to 3 acetyl residues per molecule of protein (molecular weight 170,000) for the highest enzymatic activity obtained. Both incorporation of acetyl groups into the enzyme and thiolase activity are inhibited to the same extent by iodoacetamide.The incorporation of radioactivity into the enzyme during incubation with iodo‐[1‐14C]acetamide can be divided into two kinetically different reactions: a fast incorporation with accompanying loss of enzymatic activity and a much slower process which continues after complete inactivation. From the initial rate of incorporation, it was calculated that incorporation of 3.1 carbamoylmethyl residues per molecule of protein would completely inhibit the enzyme with the highest obtained activity. This ratio compares well with the results obtained by acetyl binding studies and indicates a minimum of 3 active sites per molecule of enzyme.Enzyme labelled with iodo‐[1‐14C]acetamide was hydrolyzed with 6 N HCl. Approximately 95% of the radioactivity in the hydrolysate was found to coincide with S‐carboxymethyl‐l‐cysteine on electrophoresis and paper chromatography. Repeated crystallization with authentic carboxymethyl‐cysteine did not result in a decrease of specific radioactivity.