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m is not limited by the Affiliation amount constant to the development of your NagA-substrate sophisticated. These success (along with the improved amount of response of compound six relative to compound one) are only consistent with the summary that NagA is limited by either the development or breakdown in the tetrahedral intermediate.
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Reconstitution with the apo-enzymes was carried out overnight at 4 °C ahead of elimination of unbound metal by passage by way of a PD-ten column. The PD-ten column was pre-handled with dipicolinate to remove traces of unbound metallic after which you can washed with five column volumes of metal absolutely free HEPES buffer, pH eight.0. Soon after elution, the concentration from the metallic-reconstituted enzyme was resolute by UV absorbance and also the metal content in the samples based on ICP-MS.
Web-site link naga26 unique mutants of NagA ended up made in an effort to confirm the roles of particular residues in substrate recognition and catalytic functionality. The metallic content as well as kinetic parameters for your mutants of NagA built for this investigation are presented in Desk 4.
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