The pH dependence and modification by diethyl pyrocarbonate of isocitrate lyase from Phycomyces blakesleeanus.
We determined the variation with pH of the kinetic parameters for the isocitrate cleavage reaction catalyzed by Phycomyces isocitrate lyase, with the aim of elucidating the role played by ionising amino acid residues in binding and catalysis. The log VmaxpH profile shows that the enzyme possesses two ionising groups with pK values of 6.1 and 8.3. The first group is also observed in the VmaxpH/KmpH and pKmpH profiles, so this group is involved in catalysis. The last two profiles exhibit a similar pK value of 16 on the basic side, which represents the sum of the pK values for two ionising groups with pK values that differ by less than two pH units. Diethyl pyrocarbonate inactivated isocitrate lyase from Phycomyces with a second-order rate constant of 18.58 M-1 s-1 (at pH 6.0 and 20 degrees C). The difference spectra of the modified enzyme revealed an absorption maximum at 242 nm, characteristic of N-carbethoxyhistidine isocitrate lyase. No trough at around 280 nm due to O-carbethoxytyrosine is observed. Quantification of the increase in absorbance to 242 nm due to N-carbethoxyhistidine showed that ten histidine residues/active site were modified during total inactivation. However, only one of them was essential for catalysis. Treatment of the partially inactivated enzyme with hydroxylamine led to recovery of a substantial part of the original activity. The reactivity of isocitrate lyase towards diethyl pyrocarbonate declined with pH, following a titration curve for a group of pK 6.1. The presence of substrate decreased the rate of inactivation. Data-protection analyses indicate that the reactive histidine residues are within the active site of the enzyme.
Rúa
J
J
Departamento de Bioquímica y Biología Molecular, Universidad de León, Spain.
Soler
J
J
Busto
F
F
de Arriaga
D
D
D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007522:Isocitrate Lyase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D007523:Isocitrates / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D010800:Phycomyces / Q000201:enzymology*; D014357:Trypsin
D004791:Enzyme Inhibitors; D007523:Isocitrates; D010446:Peptide Fragments; D006639:Histidine; C034219:isocitric acid; D014357:Trypsin; D007522:Isocitrate Lyase; D008274:Magnesium; D004047:Diethyl Pyrocarbonate