The Active Site Cysteines of Thiolase

Abstract

Inactivation of thiolase with iodo [l-14C]acetamide has been shown to be due to its reaction with cysteine residues in the enzyme. [ 1 -14C]Carbamoylmethylcysteine was identified as the only product of the reaction and a study of the radioactive peptides that were isolated from a tryptic digest of the [i -14C]carbamoylmethyl-enzyme has shown that the reactive cysteines occur in a unique sequence extending to at least 26 amino acid residues in the primary structure.The enzyme-substrate compound formed when thiolase reacts with [ 1 -14C]acetyl-CoA is shown to contain [14C]acetyl groups bound in thioester linkage to a t least three cysteines per mole. Moreover these residues of [Wlacetyl-cysteine occur exclusively in a sequence that is iden tical to the sequence around the [14C]carbamoylmethylcysteine residues in the enzyme-inhibitor compound, showing that the substrate and the inhibitior react with the same four cysteine residues in the enzyme.These results provide additional evidence for the chemical identity of the four subunits of thiolase.Thiolase from pig heart is specifically inhibited by iodoacetamide and inhibition of enzyme activity is known to be due to its reaction with a t least three out of a total of 20 residues of cysteine per mole (170000 g) of enzyme protein [1,2]. Moreover when the enzyme-inhibitor compound formed by reaction of the enzyme with i~do-[~~C]acetamide was digested with trypsin three distinct radioactive peptides were found to be present by a combination of ion-exchange chromatography and paper electrophoresis [2a]. A t first sight this result could be taken to indicate that the active-site cysteines occur in different sequences in the primary structure of the enzyme. Such a conclusion, however, is not compatible with the results of the peptide mapping studies described in the preceding paper [3]. We have, therefore, reinvestigated the chemical nature of the enzyme-inhibitor compound formed when thiolase is allowed to react with iodo-[14C]acetamide. The three radioactive tryptic peptides have been isolated in pure form and amino acid sequence studies have revealed that they are chemically related and that they form part of a unique sequence around one particular cysteine residue in the primary structure of the enzyme. The reaction of the enzyme with the substrate acetyl-CoA has also been investigated. The acetyl groups that are bound to the enzyme during the enzyme catalysed reaction are shown to be bound in thioester linkage t o cysteine residues, which are moreover the same cysteine residues as were found to react with the inhibitor iodoacetamide.A preliminary account of this work has been published elsewhere [4]. MATERIALS AND METHODS[14C]Acetyl Derivatives Thiolase was prepared from pig heart as previously described 111. The enzyme (35 mg, 0.2 pmoles) was reacted with iodo-[ l-14C]acetamide (2 pmoles, 5.0 mC/mmole) in 0.06 M potassium phosphate pH 7.0 (8.5 ml) at 0". After 30 min 99Oi0 of the original enzyme activity was lost and the reaction was terminated by addition of 3 mmole...

Thiolase: A Versatile Biocatalyst Employing Coenzyme A–Thioester Chemistry for Making and Breaking C–C Bonds

“…In the biosynthetic reaction, the substrates of thiolase are two molecules of acetyl-CoA, and in the degradative reaction the substrates are 3-ketoacyl-CoA and CoA. Early studies established the importance of a nucleophilic cysteine in the active site of thiolase (12), and in either direction, the catalyzed reaction is a two-step reaction with a covalently acylated cysteine intermediate. Several spectrophotometric assays are in use to study the enzymological properties of thiolases (Supplemental Material Section 1).…”

Structural and Biochemical Studies of Substrate Selectivity in Ascaris suum Thiolases

“…The first group consists of thiolases with broad activity over a range of 3-oxoacyl-CoA carbon chain lengths (Thiolase I, EC:2.3.1.16), whereas the second group comprises short-chain thiolases that synthesize or degrade 3-oxobutyryl-CoA (Thiolase II, EC:2.3.1.9) (Scheme A). Their structure and mechanism have been investigated extensively from the initial studies of porcine heart thiolase − to those of bacterial − and human − enzymes. In the biosynthetic direction, C–C bond formation has been determined to require two catalytic Cys residues ,, and two oxyanion holes (OAHs) ,, that participate in a two-step process involving acyl–enzyme formation with the first acyl-CoA substrate followed by Claisen condensation with the second acyl-CoA substrate (Scheme B).…”