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Refined 2.5 A structure of murine adenosine deaminase at pH 6.0
A J Sharff1, D K Wilson, Z Chang
1Howard Hughes Medical Institute, Houston, TX.
Abstract:
The X-ray structure of murine adenosine deaminase complexed with the transition-state analogue 6-hydroxyl-1,6-dihydropurine ribonucleoside has been determined from a single crystal grown at pH 4.2 and transferred to mother liquor of increasing pH up to a final pH of 6.0 prior to data collection. The structure has been refined to 2.5 A to a final crystallographic R-factor of 20% using phases from the previously refined 2.4 A structure at pH 4.2. Kinetic measurements show that the enzyme is only 20% active at pH 4.2 whereas it is fully active between pH 6.0 and pH 8.5. The refined structures at either pH are essentially the same. Consideration of the pKa values of the key catalytic residues and the mechanism proposed on the basis of the structure suggests that the ionization state of these residues is largely responsible for the pH dependence on activity.
Insights
The X-ray structure of adenosine deaminase reveals its transition-state complex. Enzyme activity is pH-dependent due to catalytic residue ionization states.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Adenosine deaminase (ADA) is a crucial enzyme in purine metabolism.
- Understanding ADA's catalytic mechanism and pH dependence is vital for therapeutic targeting.
Purpose of the Study:
- To elucidate the structural basis of adenosine deaminase's pH-dependent activity.
- To characterize the X-ray structure of the murine ADA-transition-state analogue complex across a pH range.
Main Methods:
- X-ray crystallography was employed to determine the enzyme's structure.
- Crystals were grown at pH 4.2 and transferred to pH 6.0 for data collection.
- The structure was refined to 2.5 Å resolution.
Main Results:
- The refined structures at pH 4.2 and pH 6.0 were nearly identical.
- Kinetic assays demonstrated significantly reduced ADA activity at pH 4.2 compared to pH 6.0-8.5.
- The ionization states of key catalytic residues were identified as critical for pH-dependent activity.
Conclusions:
- The structural integrity of adenosine deaminase is maintained across the studied pH range.
- The pH-dependent activity of ADA is primarily attributed to the ionization state of its catalytic residues.
- This finding provides insights into ADA's catalytic mechanism and potential drug development strategies.