Purification and characterization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Penicillium chrysogenum.
delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) from Penicillium chrysogenum was purified to homogeneity by a combination of (NH4)2SO4 precipitation, protamine sulphate treatment, ion-exchange chromatography, gel filtration and hydrophobic interaction chromatography. The molecular mass of ACVS was estimated with native gradient gel electrophoresis and SDS/PAGE. The native enzyme consisted of a single polymer chain with an estimated molecular mass of 470 kDa. The denatured enzyme had an estimated molecular mass of 440 kDa. The influence of different reaction parameters such as substrates, cofactors and pH on the activity of the purified ACVS was investigated. The Km values for the three precursor substrates L-alpha-aminoadipic acid, L-cysteine and L-valine were determined as 45, 80 and 80 microM respectively, and the optimal assay concentration of ATP was found to be 5 mM (with 20 mM MgCl2). The dimer of the reaction product bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (bisACV) gave feedback inhibition of the purified ACVS; the inhibition parameter KbisACV was determined as 1.4 mM. Furthermore dithiothreitol was shown to inhibit the purified ACVS. From the addition of a glucose pulse to a steady-state glucose-limited continuous culture of P. chrysogenum it was found that there is glucose repression of the synthesis of ACVS and that there must be a constant turnover of ACVS owing to synthesis and degradation.
Theilgaard
H B
HB
Center for Process Biotechnology, Department of Biotechnology, Technical University of Denmark, DK-2800 Lyngby, Denmark.
Kristiansen
K N
KN
Henriksen
C M
CM
Nielsen
J
J
D015074:2-Aminoadipic Acid / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009842:Oligopeptides / Q000494:pharmacology; D010408:Penicillium chrysogenum / Q000201:enzymology*; D010453:Peptide Synthases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D011487:Protein Conformation; D014633:Valine / Q000378:metabolism
D004220:Disulfides; D004791:Enzyme Inhibitors; D009842:Oligopeptides; D015074:2-Aminoadipic Acid; C011638:5-(2-aminoadipyl)cysteinylvaline; D000255:Adenosine Triphosphate; D010453:Peptide Synthases; C095368:alpha-aminoadipyl-cysteinyl-valine synthetase; D014633:Valine; D008274:Magnesium; D005947:Glucose; D003545:Cysteine; D004229:Dithiothreitol