Zinc interactions with regulatory dimers from Escherichia coli aspartate transcarbamoylase

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Abstract
Zn2+ is tetrahedrally bonded to the 4 nonadjacent thiols of each regulatory chain (Mr 17 000) near r-c contacts between catalytic (c) and regulatory chains (r) in aspartate transcarbamoylase (ATCase; c6r6). This paper reports on Zn2+ interactions with r dimer in the absence of stabilizing r-c contacts. After r2 and c3 subunits were separated, -SH groups of r2 were titrated with p-(hydroxymercuri)benzenesulfonate (PMPS) at pH 7.0. The concomitant release of Zn2+ (2 equiv/r dimer) was quantitated with 4-(2-pyridylazo)resorcinol (PAR) and was a linear function of PMPS added until 8 mercaptide bonds per r2 were formed. Breakage of 1 of 4 Zn2+-sulfur bonds in a Zn2+ binding cluster therefore makes the other three bonds more labile. From stopped-flow measurements, the PMPS-promoted Zn2+ release from r2 or mercaptide bond formation with 10- to 20-fold excess PMPS/r2-SH at pH 7.0 was first order with an Arrhenius activation energy Ea = 10 kcal/mol and a half-time t1/2 = 9 .+-. 2 ms at 20.degree.C without inhibitory anions present. The rate of mercurial-promoted Zn2+ release from r2 is at least 77 times faster than that from intact c6r6 [Hunt, J. B., Neece, S. H., Schachmann, H. K., and Ginsburg, A. (1984) J. Biol. Chem. 259, 14793]; this indicates that Zn2+ binding clusters are more accessible to attack by PMPS than are those in ATCase. The addition of a 25-fold excess of the multidentate fluorescent chelator guin-2 to r2 gave a rate of Zn2+ dissociation that was 1/210th of that observed with excess mercurial. Furthermore, the Zn(PAR)1 complex was identified as the active species in the transfer of Zn2+ from Zn(PAR)2 to aporegulatory subunits, with k = (8.+-.3) .times. 105 M-1 s-1 at pH 7.0 and 15.degree.C for this second-order association reaction. Although kinetic results are dependent on the mechanisms involved, an affinity constant K''A = (1.3.+-.0.6) .times. 1012 M-1 for Zn2+ binding to r dimer at pH 7.0 and 20.degree.C in the absence and presence of 100 mM KCl could be determined spectrally by rapid equilibration with the high-affinity, sensitive metalloindicators indo-1 and quin-2. This K''A value is based on the assumptions that Zn2+ binding sites in r2 are equivalent (noninteracting) and the apo-r2 does not dissociate; if apo-r2 dissociates, K''A.apprxeq.1014 M-1. Within experimental error, the K''A value was independent of [indo-1]/[r2] ratios from 36 to 3 with 0.3-8 .mu.M r2. Thus, .DELTA.G'' .simeq. -16 kcal/mol at 20.degree.C (pH 7.0) for sequential formation of 4 Zn2+-thiol bonds (inclusive of energy changes for polypeptide chain transitions). CTP (1 mM .+-. 1 mM Mg2+) and Mg.cntdot.ATP (1 mM) increased K''A .ltoreq. .apprx. 1.2- and .apprx. 1.7-fold, respectively, for r2 binding Zn2+.