The Role of Zinc and the Reactivity of Cysteines in Escherichia coli Primase

Abstract

Primase is the zinc metalloenzyme responsible for synthesizing RNA primers for use during DNA synthesis. To establish whether the zinc played a catalytic or structural role, the zinc was removed and the activity of the apoprimase determined. The zinc was removed with p-(hydroxymercuri)-benzenesulfonate (PMPS), which covalently reacts with cysteine sulfhydryls, EDTA was added to chelate the zinc, DTT was added to remove the PMPS from the apoprimase, and then the apoprimase was separated from the small molecules. The resulting apoprimase was fully active, indicating that the zinc played a structural role but not one involved in thermodynamic folding/unfolding. PMPS and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) cysteine reactivities indicated that the cysteines fell into three categories: one or two were fast-reacting, three were zinc-ligating, and two or three were slow or nonreacting. The major distinction between apoprimase and natural primase was that apoprimase became inactivated during storage at 4 degrees C for 10 days. Storage-induced inactivation correlated with disulfide bond formation and could be reversed by incubation with a mild reducing agent. Apoprimase oxidation also prevented zinc reconstitution which was only achieved with freshly-reduced enzyme, indicating that the zinc-ligating cysteines participated in the inactivating disulfide bonds. The conclusion was that, in natural primase, the zinc prevented disulfide bond formation which, in turn, prevented inactivation. The zinc reconstitution studies identified a strong and a weak zinc binding site. Zinc could be prevented from binding to the weak site by the presence of magnesium, indicating that the weak site may be the catalytic magnesium site in which two of the seven cysteines were located.

Mercury Inactivates Transcription and the Generalized Transcription Factor TFB in the Archaeon Sulfolobus solfataricus

“…Since TFB has two cysteines in its N-terminal noncanonical zinc finger motif (responsible for recruiting RNAP) and has one cysteine in the C-terminal region (the region for binding to TBP and the promoter), it was postulated that TFB inactivation could be a result of zinc release from the zinc finger motif; however, zinc release was not observed by 4-(pyridylazo)resorcinol assay (23), a method that had been used earlier for zinc finger proteins (17). It remains possible that the lack of zinc release could be due to the fact that the cysteines in the zinc finger motif were inaccessible, perhaps reflecting unusual structural stability of this thermostable protein; however, the lack of zinc release precluded definitive conclusions regarding the site of TFB inactivation.…”

Role of Bound Zinc in Dimer Stabilization but Not Enzyme Activity of Neuronal Nitric-oxide Synthase

“…Release of Zinc by PMPS-PMPS is a mercurial reagent that can reversibly oxidize thiol groups to mercaptides, in the process releasing metals bound to the thiols (24,25,32). The amounts of zinc it released from NOS, as detected by the PAR assay (ϳ0.5 mol/mol of NOS), were greater than those detected by gel filtration/ICP-MS of thiol-free NOS samples (ϳ0.3 mol/ mol of NOS).…”

The role of the 6 lysines and the terminal amine of escherichia coli single‐strand binding protein in its binding of single‐stranded DNA

“…M13Gori ssDNA, a chimeric ssDNAconsisting of a 2,216-nucleotide G4 complementary strand origin inserted into a 6,407-nucleotide MI3 ssDNA, was purified from M13 phage as previously described (Griep & Lokey, 1996). The concentration of nucleotides in M13Gori was determined by UV spectroscopy using an extinc-tion coefficient at 260 nm of 7,370 M" cm" (Berkowitz & Day,197 …”