activation of the RNA/polymerase complex by the addition of substrate and Mg2+ initiates a single round of reaction within the crystal to form a dead-end complex that partially collapses within the enzyme active site
using Mg2+ as the divalent cation cofactor, the enzyme can use both the first conformational-change step and the phosphoryl-transfer step to distinguish between correct and incorrect nucleotides
the enzyme requires either Mn2+ or Mg2+ for RNA-dependent RNA polymerase activity. NS5B undergoes conformational changes upon the binding of metal ions. This process does not significantly stimulate the binding to the RNA or NTP substrates
the enzyme directs incoming nucleotides to its active site through Mg2+-dependent dynamics within its F motif. Mg2+-bound nucleotide first binds next to the tunnel entry of the RNA-dependent RNA polymerase(RdRp)-specific entry tunnel. Interactions with the triphosphate moiety orient the nucleotide such that its base moiety enters first. Dynamics of RdRp motifs F1 + F3 then allow the nucleotide to interrogate the RNA template base prior to nucleotide insertion into the active site. These dynamics are finely regulated by a second magnesium dication, thus coordinating the entry of a magnesium-bound nucleotide with shuttling of the second magnesium necessary for the two-metal ion catalysis
in presence of Mg2+ significant activity is observed when poly(A) or poly(C) is used as template and the activity is template and primer-dependent. Poly(G) and poly(U) templates are not efficient substrates. Biotinylated oligoDNA primers appear to work slightly more efficiently than oligoRNA primers. Divalent cation required, optimal concentration for Mg2+ is 1 mM
using Mn2+ as the cofactor, the ability to diminish the rate of phosphoryl transfer for incorrect nucleotides relative to correct nucleotides is lost completely, leaving only the conformational-change step for selection of the correct nucleotide
the enzyme requires either Mn2+ or Mg2+ for RNA-dependent RNA polymerase activity. NS5B undergoes conformational changes upon the binding of metal ions. This process does not significantly stimulate the binding to the RNA or NTP substrates
required, GTP binding reduces the affinity of jRdRp for RNA in the absence of Mn2+, binding affinity of jRdRp toward RNA in the presence of GTP/ATP is restored when Mn2+ ion is included in the reaction buffer
in presence of Mn2+ activity is stimulates by 2.5-5.6fold. RNA synthesis using poly(C) as template becomes primer-independent, about 2.5fold stimulation at 1 mM
0.05 mM, 40% of the activity compared to reaction with 3 mM Mg2+. 5.0 mM, 70% of the activity compared to the reaction with 3 mM Mg2+, Mn2+ is present as the sole divalent cation
the study models potential Zn2+ binding to study models potential Zn binding to the enzyme (RdRp) and the 3CLpro. The Zn binding site is conserved between severe acute respiratory syndrome (SARS)-coronavirus (CoV) and SARS-CoV-2. The location of these sites may influence the enzymatic activity of the enzyme in COVID-19
computational study of metal-binding preferences. When he calculated metal hydration energies are used, the proteination energies for the structures indicate that the binding preference for the metal is Cu2+ > Mn2+, Fe2+ > Zn2+ > Ni2+ > Co2+ > Mg2+, Ca2+