No abstract

No abstract

Serine sulphhydrase from chicken liver and cysteine lyase from chicken-embryo yolk sac catalyse the exchange of alpha-H atoms of the amino acid substrate with 3-H-2O. The degree of labelling of the unreacted substrate approaches a maximum of one atom per mol of amino acid. In the absence of replacing agent there is practically no H-exchange in the substrate. The alpha-H of the accumulating beta-substitution product is completely replaced by the labelled hydrogen of the solvent water, irrespective of the duration of incubation. The amount of labelled alpha-hydrogen incorporated into excess (unreacted) amino acids substrate within 3.5-h incubation is somewhat less than the amount incorporated into the product of the complete enzymic beta-replacement reaction. Within the sensitivity limits of detection, the enzymes do not induce any isotopic exchange either of b-H atoms in the amino substrate or of 18-O-labelled beta-HO groups, in the case of L-serine. Neither serine sulphhydrase nor cysteine lyase will catalyse alpha-hydrogen exchange in close structural analogues of their substrates, e.g. L-alanine, D-serine, threonin, 3-phosphoserine. A special case is the interaction of cysteine lyase with the competitive inhibitor, L-serine (whose inhibitor constant, K-i, is equal to the Michaelis constant, K-m, of L-cysteine): the lyase catalyses, only in presence of a cosubstrate thiol, alpha-H exchange in L-serine at approximately the same rate as in L-cysteine. The present data concerning isotopic alpha-H exchange in substrate amino acids, and evidence published earlier, suggest that the catalytic mechanism of replacement-specific beta-lyases may significantly differ from that of the eliminating or ambivalent (mixed-function) lyases. Formation of alpha, beta-unsaturated pyridoxylidene aldimines as real reaction intermediates is unlikely in the case of lyases specifically catalysing beta-replacement reactions; these may proceed by some alternative mechanism of the type suggested in this paper.

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...

Compounds of the general formula RONH 2 containing structural fragments of the products or the substrates of enzymatic reactions are suggested as· a means for effective and specific inhibition of corresponding carbonyl dependent enzymes.