These tools will no longer be maintained as of December 31, 2024. Archived website can be found here. PubMed4Hh GitHub repository can be found here. Contact NLM Customer Service if you have questions.


Pubmed for Handhelds

PUBMED FOR HANDHELDS

Search MEDLINE/PubMed

  • Title: Isocitrate lyase from Cephalosporium acremonium. Role of Mg2+ ions, kinetics, and evidence for a histidine residue in the active site of the enzyme.
    Author: Perdiguero E, de Arriaga D, Busto F, Soler J.
    Journal: Biochemistry; 1995 May 09; 34(18):6059-68. PubMed ID: 7742309.
    Abstract:
    Isocitrate lyase was purified from Cephalosporium acremonium CW-19 from cultures growing with poly(oxyethylene)sorbitan monopalmitate as the carbon source. Its subunit M(r) and native M(r) were 63,000 +/- 2000 and 250,000 +/- 5000, respectively. We found the Mg(2+)-isocitrate complex to be the true substrate and that Mg2+ ions act as a nonessential activator, according to the model reported by Giachetti et al. (1988) [Giachetti, E., Pinzauti, G., Bonaccorsi, R., & Vanni, P. (1988) Eur. J. Biochem. 172, 85-91], from which the kinetic parameters were calculated. The kinetic study is consistent with an ordered Uni-Bi mechanism, and the kinetic and rate constants of the model were calculated. pH dependence of the cleavage reaction indicated that the catalysis was dependent on two dissociable groups on the enzyme-substrate complex. The enzyme was inactivated by diethyl pyrocarbonate following first-order kinetics at all reagent concentrations used. The pseudo-first-order rate constant of inactivation increases with pH, suggesting participation of an amino acid residue with pK 6.0. Hydroxylamine added to the inactivated enzyme quickly restored the incremental absorption at 240 nm and most of the activity. Data analyses indicated that diethyl pyrocarbonate inactivation is a consequence of modification of 11 histidine residues per enzyme subunit, and from statistical analysis, we concluded that one is catalytically important. Mg(2+)-isocitrate protects the enzyme against diethyl pyrocarbonate inactivation with a Ks value of 26.8 +/- 2.1 microM, close to the Km value. Isocitrate protects the enzyme but a high concentration, suggesting its binding to the catalytic site of the nonactivated enzyme. Mg2+ ions also produced total competitive protection.
    [Abstract] [Full Text] [Related] [New Search]