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A still unknown low-molecular-mass cofactor essential for the activity of carnitine-metabolizing enzymes (e.g., L-carnitine dehydratase, crotonobetaine reductase) from E. coli has been purified to homogeneity from a cell-free extract of E. coli O44K74. The purity of the cofactor was confirmed by HPLC analysis. Biosynthesis of the unknown compound was only observed when bacteria were cultivated anaerobically in the presence of L-carnitine or crotonobetaine. The determined properties, together with results obtained from UV-visible, (1)H NMR, and mass spectrometry, indicate that the compound in question is a new CoA derivative. The esterified compound was suggested to be gamma-butyrobetaine-a metabolite of carnitine metabolism of E. coli. Proof of structure was performed by chemical synthesis. Besides gamma-butyrobetainyl-CoA, a second new CoA derivative, crotonobetainyl-CoA, was also chemically synthesized. Both CoA derivatives were purified and their structures confirmed using NMR and mass spectrometry. Comparisons of structural data and of the chemical properties of gamma-butyrobetainyl-CoA, crotonobetainyl-CoA, and the isolated cofactor verified that the unknown compound is gamma-butyrobetainyl-CoA. The physical and chemical properties of gamma-butyrobetainyl-CoA and crotonobetainyl-CoA are similar to known CoA derivatives.

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Phosphofructokinase-1 (Pfk-1) from Schizosaccharomyces pombe was purified by 54-fold enrichment to homogeneity elaborating the following steps: (a) Disruption of the cells with glass beads; (b) fractionated precipitation with polyethylene glycol 6000; (c) affinity chromatography on Cibacron-Blue F3G-A-Sephadex G 100; (d) ion exchange chromatography on Resource Q. The native enzyme exhibits a mass of 790+/-30 kDa, as detected by sedimentation equilibrium measurements. The apparent sedimentation coefficient was found to be s(20,c)=20.2+/-0.3 S. No significant dependence of the s-value on the protein concentration was observed in the range 0. 07-0.7 mg/ml. Polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate and MALDI-TOF spectra showed that the enzyme is composed of subunits of identical size of 100+/-5 kDa, forming an octameric structure. The N-terminus of the enzyme was found to be blocked. Sequences of tryptic and chymotryptic peptides of the subunit coincide with the proposed amino acid sequence as deduced from the gene from the EMBL library. The Pfk-1 coding sequence of S. pombe was transformed into a Pfk-1 double deletion mutants of Saccharomyces cerevisiae resulting in glucose-positive cells with enzyme activity in the crude cell extract. The kinetic analysis revealed less cooperativity to fructose 6-phosphate (n(H)=1.6) and less inhibition by ATP as compared to the enzyme from baker's yeast. Fructose 2,6-bisphosphate (in micromolar range) and AMP (in millimolar range) were found to overcome ATP inhibition and to increase the affinity to fructose 6-phosphate.