A Convenient gHMQC-Based NMR Assay for Investigating Ammonia Channeling in Glutamine-Dependent Amidotransferases: Studies of Escherichia coli Asparagine Synthetase B

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• 作者：Kai K. Li ; William T. Beeson ; IV ; Ion Ghiviriga ; Nigel G. J. Richards
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：April 24, 2007
• 年：2007
• 卷：46
• 期：16
• 页码：4840 - 4849
• 全文大小：292K
• 年卷期：v.46,no.16(April 24, 2007)
• ISSN：1520-4995

文摘

X-ray crystal structures of glutamine-dependent amidotransferases in their "active" conformationhave revealed the existence of multiple active sites linked by solvent inaccessible intramolecular channels,giving rise to the widely accepted view that ammonia released in a glutaminase site is channeled efficientlyinto a separate synthetase site where it undergoes further reaction. We now report a very convenientisotope-edited ¹H NMR-based assay that can be used to probe the transfer of ammonia between the activesites of amidotransferases and demonstrate its use in studies of Escherichia coli asparagine synthetase B(AS-B). Our NMR results suggest that (i) high glutamine concentrations do not suppress ammonia-dependent asparagine formation in this bacterial asparagine synthetase and (ii) ammonia in bulk solutioncan react with the thioester intermediate formed during the glutaminase half-reaction by accessing theN-terminal active site of AS-B during catalytic turnover. These observations are consistent with a modelin which exogenous ammonia can access the intramolecular tunnel in AS-B during glutamine-dependentasparagine synthesis, in contrast to expectations based on studies of class I amidotransferases.