Relation of enzyme activity to local/global stability of murine adenosine deaminase: 19F NMR studies.


Abstract

Adenosine deaminase (ADA, EC 3.5.4.4) 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

Submission Details

ID: qVQ54BdG

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:50 p.m.

Version: 1

Publication Details
Shu Q;Frieden C,J. Mol. Biol. (2005) Relation of enzyme activity to local/global stability of murine adenosine deaminase: 19F NMR studies. PMID:15581901
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
3MVI 2010-11-03 1.6 Crystal structure of holo mADA at 1.6 A resolution
1A4M 1998-10-14 1.95 ADA STRUCTURE COMPLEXED WITH PURINE RIBOSIDE AT PH 7.0
3KM8 2010-10-20 2.0 Crystal structuore of adenosine deaminase from mus musculus complexed with 9-deazainosine
3MVT 2010-10-13 2.2 Crystal structure of apo mADA at 2.2A resolution
3T1G 2012-02-08 2.35 Engineering of organophosphate hydrolase by computational design and directed evolution
1FKX 1996-08-01 2.4 MURINE ADENOSINE DEAMINASE (D296A)
1UIP 1997-06-24 2.4 ADENOSINE DEAMINASE (HIS 238 GLU MUTANT)
2ADA 1995-03-31 2.4 ATOMIC STRUCTURE OF ADENOSINE DEAMINASE COMPLEXED WITH A TRANSITION-STATE ANALOG: UNDERSTANDING CATALYSIS AND IMMUNODEFICIENCY MUTATIONS
1UIO 1997-06-24 2.4 ADENOSINE DEAMINASE (HIS 238 ALA MUTANT)
1FKW 1996-08-01 2.4 MURINE ADENOSINE DEAMINASE (D295E)
1ADD 1994-01-31 2.4 A PRE-TRANSITION STATE MIMIC OF AN ENZYME: X-RAY STRUCTURE OF ADENOSINE DEAMINASE WITH BOUND 1-DEAZA-ADENOSINE AND ZINC-ACTIVATED WATER
1A4L 1998-10-14 2.6 ADA STRUCTURE COMPLEXED WITH DEOXYCOFORMYCIN AT PH 7.0

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
94.0 Adenosine deaminase Q920P6 ADA_RAT
100.0 Adenosine deaminase P03958 ADA_MOUSE