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Specificity and pH dependence for acylproline cleavage by prolidase
W L Mock1, P C Green, K D Boyer
1Department of Chemistry, University of Illinois, Chicago 60680-4348.
The Journal of Biological Chemistry
|November 15, 1990
Summary
The enzyme prolidase activity is pH-dependent, with a key residue at pH 6.6. A reverse-protonation mechanism involving substrate binding and a metal ion is proposed for its hydrolytic function.
Area of Science:
- Enzymology
- Biochemistry
- Protein Catalysis
Background:
- Prolidase is a metalloenzyme crucial for collagen processing.
- Understanding its catalytic mechanism and pH dependence is vital for biochemical studies.
Purpose of the Study:
- To investigate the pH dependence of prolidase hydrolytic activity.
- To elucidate the role of specific residues and substrate interactions in catalysis.
Main Methods:
- Enzymatic assays with dipeptide substrates.
- Kinetic analysis (kcat/Km and kcat) across a pH range.
- Studies with modified substrate analogs.
Main Results:
- Catalytic activity showed bell-shaped (kcat/Km) or sigmoidal (kcat) pH dependence.
- An active site residue with a pKa of 6.6 was critical.
- Substrate amino group and a Lewis-acidic metal ion interaction were implicated.
Conclusions:
- A reverse-protonation mechanism for substrate binding and activation is proposed.
- The findings provide insights into prolidase's catalytic strategy and metal ion coordination.