LysK, the enzyme lysing Staphylococcus aureus cells: specific kinetic features and approaches towards stabilization.
LysK, the enzyme lysing cells of Staphylococcus aureus, can be considered as perspective antimicrobial agent. Knowledge of LysK properties and behavior would allow optimizing conditions of its storage as well as formulating strategy towards its stabilization. Reaction of LysK with substrate (suspension of autoclaved Staphylococcus aureus cells) has been found to be adequately described by the two-stage Michaelis-Menten kinetic scheme. Ionization of the enzyme and enzyme-substrate complex is important for revealing catalytic activity, which is controlled by two ionogenic groups with pK 6.0 and 9.6. Ionization energy of the group with pK 6.0 is of 30 kJ/mol, thus, pointing out on His residue; pK 9.6 might be attributed to metal ion or metal-bound water molecule. At temperatures lower than 40 degrees C, LysK stability depends on its concentration, pH and presence of low molecular weight additives. Results of electrophoresis under native and denaturing conditions as well as sedimentation analysis strongly suggest that aggregation is behind LysK inactivation. Decrease in the enzyme concentration, as well as addition of low molecular mass polyols (glycerol, sorbitol, sucrose, trehalose) and Ca(2+) cations resulted in an enhanced (more than 100 times) stability of LysK. Dramatic stability decline observed in a narrow temperature range (40-42 degrees C) was accompanied by changes in LysK secondary structure as confirmed by CD spectroscopy studies. According to computer modeling data, Cys and His residues and metal cation might play a crucial role for LysK catalytic activity. Our data on the enzyme activity in the presence of ethylenediaminetetraacetic acid and different metal cations confirmed the importance of metal cation in LysK catalysis.
Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Vorobievy Gory, 1-11, Moscow 119991, Russia.
Lyubov Yu
LY
Filatova
Stephen C
SC
Becker
David M
DM
Donovan
Alexander K
AK
Gladilin
Natalia L
NL
Klyachko
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D013211:Staphylococcus aureus / Q000166:cytology*
D001426:Bacterial Proteins; D000581:Amidohydrolases