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Updated: May 9, 2026
Quantitative Methods to Study Protein Arginine Methyltransferase 1-9 Activity in Cells
Published on: August 7, 2021
Kinetic characterization of arginine deiminase and carbamate kinase from Streptococcus pyogenes M49
Silvio Hering1, Antje Sieg, Bernd Kreikemeyer
1Rostock University Medical Centre, Institute of Medical Microbiology, Virology, and Hygiene, Schillingallee 70, 18057 Rostock, Germany.
Abstract:
Streptococcus pyogenes (group A Streptococcus, GAS) is an important human pathogen causing mild superficial infections of skin and mucous membranes, but also life-threatening systemic diseases. S. pyogenes and other prokaryotic organisms use the arginine deiminase system (ADS) for survival in acidic environments. In this study, the arginine deiminase (AD), and carbamate kinase (CK) from S. pyogenes M49 strain 591 were heterologously expressed in Escherichia coli DH5α, purified, and kinetically characterized. AD and CK from S. pyogenes M49 share high amino acid sequence similarity with the respective enzymes from Lactococcus lactis subsp. lactis IL1403 (45.6% and 53.5% identical amino acids) and Enterococcus faecalis V583 (66.8% and 66.8% identical amino acids). We found that the arginine deiminase of S. pyogenes is not allosterically regulated by the intermediates and products of the arginine degradation (e.g., ATP, citrulline, carbamoyl phosphate). The Km and Vmax values for arginine were 1.13±0.12mM (mean±SD) and 1.51±0.07μmol/min/mg protein. The carbamate kinase is inhibited by ATP but unaffected by arginine and citrulline. The Km and Vmax values for ADP were 0.72±0.08mM and 1.10±0.10μmol/min/mg protein and the Km for carbamoyl phosphate was 0.65±0.07mM. The optimum pH and temperature for both enzymes were 6.5 and 37°C, respectively.
Insights
Streptococcus pyogenes uses the arginine deiminase system (ADS) for survival. This study characterized key enzymes arginine deiminase (AD) and carbamate kinase (CK), revealing their kinetic properties and optimal conditions for function.
Area of Science:
- Microbiology
- Enzymology
- Biochemistry
Background:
- Streptococcus pyogenes (group A Streptococcus, GAS) is a significant human pathogen.
- GAS utilizes the arginine deiminase system (ADS) for survival in acidic environments.
- Understanding GAS enzymes involved in ADS is crucial for pathogen research.
Purpose of the Study:
- To heterologously express, purify, and kinetically characterize arginine deiminase (AD) and carbamate kinase (CK) from S. pyogenes M49.
- To compare S. pyogenes AD and CK with homologous enzymes from other bacteria.
- To investigate the regulatory mechanisms of S. pyogenes AD and CK.
Main Methods:
- Heterologous expression of S. pyogenes M49 arginine deiminase (AD) and carbamate kinase (CK) in E. coli DH5α.
- Purification of recombinant AD and CK enzymes.
- Kinetic characterization including determination of Km, Vmax, and optimal pH/temperature, and assessment of allosteric regulation and inhibition.
Main Results:
- S. pyogenes AD and CK share sequence similarity with orthologs from Lactococcus lactis and Enterococcus faecalis.
- Arginine deiminase (AD) is not allosterically regulated by ADS intermediates or products.
- Carbamate kinase (CK) is inhibited by ATP but not by arginine or citrulline; kinetic parameters for ADP and carbamoyl phosphate were determined.
- Optimal pH and temperature for both enzymes were found to be 6.5 and 37°C, respectively.
Conclusions:
- The arginine deiminase system enzymes from S. pyogenes have distinct kinetic properties.
- The lack of allosteric regulation in S. pyogenes AD suggests a simplified regulatory mechanism compared to some other bacteria.
- Kinetic data provides a foundation for understanding the role of ADS in S. pyogenes pathogenesis and survival.
