Interaction of acetyl‐CoA fragments with rat liver acetyl‐CoA carboxylase

Abstract

The interaction of acetyl-CoA fragments with rat liver acetyl-CoA carboxylase has been studied. Dephosphorylated acetyl-CoA did not actually differ from acetyl-CoA in its substrate properties. Non-nucleotide analogues of the substrate, S-acetylpantatheine and it's 4'-phosphate, also possess substrate properties (V,,, = 1.5% and 15% of the maximal rate value of acetyl-CoA carboxylation, respectively). The nucleotide fragment in the acetyl-CoA molecule produces a marked effect on the thermodynamics of the substrate-enzyme interaction, and is apparently involved in activation and appropriate orientation of the acetyl group in the active site. The better substrate properties of S-acetylpantetheine 4-phosphate and the inhibitory properties of pantetheine 4'-phosphate, compared to the unphosphorylated analogues, evidence an important role of the 5'-P-phosphate of 3'-phosphorylated ADP residue in acetyl-CoA binding to the enzyme.Acetyl-CoA carboxylase catalyzes the first step in the pathway of long-chain fatty acid biosynthesis and plays a critical role in the regulation of this process [l, 21. CoA modulates the enzyme activity in the cell, though binding to the specific kinase of carboxylase [3] and directly activating carboxylase at low concentration [4]. The long-chain acyl-CoA are effective competitive inhibitors of the enzyme which assist in the dissociation of the active polymeric form of the enzyme 151. A CoA precursor, pantethine, has found a wide application as a hypolipidaemic drug [6]. According to our data, the 4'-phosphate of D-pantothenic acid possesses a similar activity. Data are available featuring peculiarities of the interaction of CoA, its analogues and precursors with CoA-dependent enzymes [7 -91. However, no data have been reported yet on the specificity of acetyl-CoA carboxylase toward any CoA precursors. The aim of this work was to study the interaction of acetyl-CoA carboxylase with a successive series of CoA precursors and their S-acetyl derivatives, which would allow the evaluation of the individual contributions of different fragments of the CoA molecule to interaction with the enzyme. MATERIALS AND METHODSThe following reagents were used in the study: ATP, phenylmethylsulphonyl fluoride, EDTA from Serva; tris(hydroxymethyl) aminomethane, dithiothreitol, bovine serum albumin (fraction V), tosyl-2-phenylalaninechloromethane, tosyl-L-lysinechloromethane, D-biotin, CoASH, acetyl-CoA, cyanogen bromide purchased from Sigma; avidin from Biolar (USSR), CI-Sepharose 4B from Pharmacia, potassium Dpantothenate and potassium D-pantothenate 4'-phosphate Synthesis of acetyl-CoA and its precursorsD-Pantetheine 4-phosphate was obtained as described previously [8]. Acetyl-CoA, S-acetylpantetheine, and S-acetylpantetheine 4-phosphate were synthesized by acetylation with acetic anhydride (in the latter two cases, after reducing the corresponding disulfides by NaBH4) [lo]. S-Acetylcysteamine was prepared from 2-mercaptoethylamine hydrochloride and acetyl chloride by the procedure of Foyl et al. 1111. Enzyme p...

Coenzyme A Analogues and Derivatives: Synthesis and Applications as Mechanistic Probes of Coenzyme A Ester-Utilizing Enzymes

“…The acceptance of simplified derivatives of CoA as substrates varies greatly among different CoA-utilizing enzymes. For example, dephospho-CoA, lacking only the 3‘-phosphate group, is a very poor substrate for phosphotransacetylase, while the acetyl derivatives of CoA and dephospho-CoA are virtually indistinguishable as substrates for acetyl-CoA carboxylase . Acetylphosphopantetheine and acetylpantetheine exhibit values of V max with acetyl-CoA carboxylase of 15% and 1.5%, respectively, relative to acetyl-CoA, with K m values increased about 10- and 30-fold .…”

“…For example, dephospho-CoA, lacking only the 3‘-phosphate group, is a very poor substrate for phosphotransacetylase, while the acetyl derivatives of CoA and dephospho-CoA are virtually indistinguishable as substrates for acetyl-CoA carboxylase . Acetylphosphopantetheine and acetylpantetheine exhibit values of V max with acetyl-CoA carboxylase of 15% and 1.5%, respectively, relative to acetyl-CoA, with K m values increased about 10- and 30-fold . Pantetheine and phosphopantetheine esters are very poor substrates for crotonase, with activity less than 0.1% the activity with the natural CoA ester as substrate, though some increased activity can be induced by including AMP or better yet 3‘,5‘-ADP in the assay mixture. , On the basis of the crystal structure, this increased activity may be attributed to the hydrogen bonding of the adenine amino group to an amino acid involved directly in catalysis, a structural feature which is also observed in the p -chlorobenzoyl-CoA dehalogenase …”