Mercurial-promoted Zn2+ release from Escherichia coli aspartate transcarbamoylase.
The release of Zn2+ from aspartate transcarbamoylase (ATCase; c6r6) upon challenge by p-hydroxymercuriphenylsulfonate (PMPS) has been studied using the sensitive, high-affinity metallochromic indicator 4-(2-pyridylazo)resorcinol at pH 7.0. When the--SH group of each catalytic (c) chain is protected, 1 Zn2+ is released for every 4 eq of PMPS added to ATCase during titration of the 24--SH groups of regulatory (r) chains. Moreover, the release of Zn2+ is a linear function of PMPS added, indicating that the rate-limiting step in Zn2+ release is mercurial attack on the 1st of the 4 r--SH groups bonded tetrahedrally to Zn2+ in an r chain near c:r contacts. Dissociation of ATCase is linked to Zn2+ release and mercaptide formation; e.g. upon addition of 4 eq of PMPS to ATCase in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) buffer, 1/6th of ATCase is dissociated to c3 and r2 subunits at approximately 83% of the rate of Zn2+ release, with no accumulation of the c6r4 intermediate as is observed in KPO4 buffer. Adding less than or equal to 4 PMPS/ATCase, the release of Zn2+ is first-order in [PMPS] and is virtually independent of [ATCase] with an activation energy of 18 kcal/mol. With large excesses of PMPS, stopped-flow traces show a lag period followed by pseudo first-order release of Zn2+ from ATCase and the reaction order in [PMPS] = approximately 1.3. Under these conditions, PMPS has a chaotropic effect on ATCase; the activation energy for Zn2+ release is much lower than that obtained with limiting PMPS and is increased by the presence of phosphate or active-site ligand from 6.6 to approximately 12 kcal/mol. A reasonable explanation of the observed kinetic data is that the organomercurial reagent binds reversibly to nitrogenous side chain groups in an ATCase molecule prior to the rate-limiting reaction with a sulfhydryl group.
D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008024:Ligands; D010663:Phenylmercury Compounds / Q000494:pharmacology*; D011485:Protein Binding; D013816:Thermodynamics; D015032:Zinc / Q000378:metabolism*
D008024:Ligands; D010663:Phenylmercury Compounds; C002910:4-hydroxymercuribenzenesulfonate; D001221:Aspartate Carbamoyltransferase; D015032:Zinc