Adenosine deaminase (ADA, EC 188.8.131.52) is a ubiquitous (beta/alpha)8-barrel enzyme crucial for purine metabolism and normal immune competence. In this study, it was observed that loss of enzyme activity of murine ADA (mADA) precedes the global secondary and tertiary structure transition when the protein is exposed to denaturant. The structural mechanism for this phenomenon was probed using site-specific 19F NMR spectroscopy in combination with [6-19F]tryptophan labeling and inhibitor binding. There are four tryptophan residues in mADA and all are located more than 12 A from the catalytic site. The 19F NMR spectra of [6-19F]Trp-labelled mADA show that the urea-induced chemical shift change of 19F resonance of W161, one of the four tryptophan 19F nuclei, correlates with the loss of enzyme activity. The urea-induced chemical shift change of another 19F resonance of W117 correlates with the change of the apparent rate constant for the binding of transition-state analogue inhibitor deoxycoformycin to the enzyme. On the other hand, the chemical environment of the local region around W264 does not change significantly, as a consequence of perturbation by low concentrations of urea or substrate analog. The results indicate that different regions of mADA have different local stability, which controls the activity and stability of the enzyme. The results provide new insights into the relationship between the function of a protein and its conformational flexibility as well as its global stability. This study illustrates the advantage of 19F NMR spectroscopy in probing site-related conformational change information in ligand binding, enzymatic activity and protein folding. Study holds ProTherm entries: 18493, 18494, 18495, 18496, 18497, 18498 Extra Details: 2 mM DTT was added in the experiment (b/a)8-barrel; protein unfolding; chemical shift; substrate binding; local stability
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:50 p.m.
|Number of data points||14|
|Proteins||Adenosine deaminase ; Adenosine deaminase|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: Cm prot_conc:5 nM ; Experimental Assay: Cm prot_conc:10 microM ; Experimental Assay: m prot_conc:10 microM ; Experimental Assay: dG_H2O prot_conc:10 microM ; Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O|
|Libraries||Mutations for sequence MAQTPAFNKPKVELHVHLDGAIKPETILYFGKKRGIALPADTVEELRNIIGMDKPLSLPGFLAKFDYYMPVIAGCREAIKRIAYEFVEMKAKEGVVYVEVRYSPHLLANSKVDPMPWNQTEGDVTPDDVVDLVNQGLQEGEQAFGIKVRSILCCMRHQPSWSLEVLELCKKYNQKTVVAMDLAGDETIEGSSLFPGHVEAYEGAVKNGIHRTVHAGEVGSPEVVREAVDILKTERVGHGYHTIEDEALYNRLLKENMHFEVCPWSSYLTGAWDPKTTHAVVRFKNDKANYSLNTDDPLIFKSTLDTDYQMTKKDMGFTEEEFKRLNINAAKSSFLPEEEKKELLERLYREYQ|