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Common-type acylphosphatase: steady-state kinetics and leaving-group dependence
1Dipartimento di Scienze Biochimiche, Università di Firenze, Viale Morgagni 50, Firenze, Italy.
The Biochemical Journal
|November 14, 1997
Summary
Acylphosphatase enzyme activity is highly dependent on substrate structure, with a linear relationship observed between catalytic rate and leaving group acidity. The enzyme likely releases inorganic phosphate as the final step, without forming phosphoenzyme or acyl intermediates.
Area of Science:
- Biochemistry
- Enzymology
- Protein Catalysis
Background:
- Acylphosphatase is an enzyme that hydrolyzes acyl phosphates.
- Understanding its catalytic mechanism is crucial for enzyme kinetics and drug development.
- Previous studies have investigated substrate specificity but a detailed mechanistic insight is lacking.
Purpose of the Study:
- To synthesize various acyl phosphates with differing acyl moieties.
- To elucidate the catalytic mechanism of the bovine common-type acylphosphatase isoenzyme.
- To determine the kinetic parameters (Km, Vmax, kcat) and activation energy for substrate hydrolysis.
Main Methods:
- Synthesis of diverse acyl phosphate substrates.
- Enzymatic assays using bovine common-type acylphosphatase isoenzyme at pH 5.3 and 25°C.
- Kinetic analysis, including determination of Km, Vmax, kcat, and competitive inhibition studies with inorganic phosphate (Pi).
- Isotopic labeling studies using H218O to investigate reaction intermediates.
Main Results:
- Observed wide variations in kcat values across different acyl phosphate substrates.
- Demonstrated linear relationships between log kcat and leaving group pKa, and log kcat/Km and leaving group pKa.
- Found similar Km values for all substrates, suggesting interaction primarily with the phosphate moiety.
- Showed no transphosphorylation, H218O-inorganic phosphate exchange, or formation of phosphoenzyme or acyl intermediates.
- Identified competitive inhibition by Pi and lack of inhibition by carboxylate ions.
Conclusions:
- The catalytic rate (kcat) of acylphosphatase is strongly influenced by the leaving group's acidity.
- The enzyme's active site primarily interacts with the phosphate group of the substrate.
- The catalytic mechanism likely involves direct hydrolysis of the acyl phosphate, releasing Pi as the final step, without covalent enzyme-bound intermediates.