Role for zinc in the quaternary structure of aspartate transcarbamylase from Escherichia coli

Abstract

Aspartate transcarbamylase (ATCase) from Escherichia coli was found to contain zinc in the amount of 6 ± 1 atoms per molecule of protein (3.1 X 105 daltons). Zn2+ was not removed from the protein by prolonged dialysis against solutions of strong chelators and did not exchange detectably with free 66Zn2+ during an exposure period of 40 days. The requirement of metal ions for the formation of intact ATCase could be demonstrated in vivo. E. coli, forced to synthesize enzyme in Zn2+-deficient medium, contained only 30% of their transcarbamylase activity as ATCase, with the remaining 70 % present as catalytic subunit, whereas bacteria grown with the normal excess concentration of Zn2+ had less than 5 % of their transcarbamylase activity as the catalytic subunit. When purified ATCase was treated with the mercurial p-mercuribenzoate (PMB), dissociation into catalytic and regulatory subunits occurred and Zn2+ became easily removable from the protein by dialysis against chelators, or during the standard procedure employed for the isolation of subunits (Gerhart, J. C., and Holoubek, H. (1967), J. Biol. Chem. 242, 2881). Regulatory subunit, obtained by this procedure, contained no detectable Zn2+ but contained tightly bound mercuric ion in variable amounts ranging from 0.1 to 1.0 atom per polypeptide chain (1.7 X 104 daltons). Mercuric ion appeared to be released from PMB during subunit isolation. The mercury content of regulatory subunit preparations correlated positively with their capacity to aggregate with catalytic subunit to form reconstituted ATCase. The isolated catalytic subunit also lacked Zn2+ and, unlike the regulatory subunit, contained no detectable Hg2+. This finding indicates that the catalytic activity of the enzyme does not depend on metal ions such as zinc or mercury. A modified procedure for T X he regulatory enzyme, aspartate transcarbamylase (ATCase)1 from Escherichia coli, contains two types of protein subunits, one the catalytic subunit which possesses active

Biphasic modulation of ryanodine binding to sarcoplasmic reticulum vesicles of skeletal muscle by Zn2+ ions

“…As the second distinct feature of the effect of Zn# + , the present study found that binding in the presence of high [Zn# + ] f showed a biphasic time course (Figures 3 and 4 Recent studies have shown that Zn# + ions are important for the stability of protein structure in various cells [6,7,16,17]. Although a long polypeptide can fold into an appropriate structure autonomously, the binding of Zn# + ions is necessary for stabilizing the folded conformation of short polypeptides [7].…”

Aspartate Transcarbamoylase from Escherichia coli : Electron Density at 5.5 Å Resolution

“…The location of the Zn ion has been transferred to our electron-density map (Fig. 5), and it appears to be near the R-C interface, as has been suggested (15,16). However, it must be kept in mind that the zinc position was determined in the CTPATCase crystals, whereas the map is of unliganded ATCase, and the enzyme may have different conformations in the two crystal forms.…”

Three-dimensional structures of aspartate carbamoyltransferase from Escherichia coli and of its complex with cytidine triphosphate.

“…Molecular weights are 34,000 for C and 17,000 for R (5,8,9). Each regulatory unit contains one Zn2 , which has been presumed to coordinate to the four sulfhydryl groups of this chain (10,11), and which is necessary for reconstitution of the enzyme from the subunits C3 and R2. Other properties of this enzyme have been summarized recently (12).…”