The role of zinc and the reactivity of cysteines in Escherichia coli primase.
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.
Griep
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MA
Department of Chemistry, University of Nebraska, Lincoln 68588-0304, USA.
Lokey
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ER
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D003545:Cysteine / Q000378:metabolism*; D019915:DNA Primase; D004220:Disulfides; D004926:Escherichia coli / Q000201:enzymology; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D012313:RNA / Q000096:biosynthesis; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000378:metabolism*; D015032:Zinc / Q000032:analysis / Q000378:metabolism*
D001059:Apoproteins; D004220:Disulfides; D008667:Metalloproteins; C034477:RNA primers; D012313:RNA; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D015032:Zinc; D003545:Cysteine