Quantization of pH: evidence for acidic activity of triglyceride lipases.
Here we present a study of lipolytic activity of lipases from Fusarium solani pisi (cutinase), Rhizomucor miehei, Pseudomonas cepacia, and Humicola lanuginosa. Their activities toward triolein provide clear evidence for considerable enzymatic activity under acidic conditions. The activity was followed using Fourier transform infrared attenuated total reflection (FTIR-ATR) and nuclear magnetic resonance (NMR). Using these approaches, all the lipases that were studied exhibited lipolytic activity down to pH 4. The common model for the catalytic activity of the F. solani pisi cutinase, and lipases in general, requires the deprotonation of the active site histidine. Measurements using (13)C NMR spectroscopy showed a pK(a) value in the absence of substrate that is not consistent with the detected acid activity. We propose a novel model for the electrostatics in the active site of cutinase that could explain the observed acidic activity. The active site is essentially covered with the lipid surface during catalysis, thus preventing chemical communication between the active site and the bulk solvent. We propose that the classical definition of pH in bulk solution is not applicable to the active site environment of a lipase when the active site is inaccessible to solvent. In small restricted volumes, the pH must be quantized, and since much of the biological world is dependent on compartmentalization of processes in small volumes, it becomes relevant to investigate when this mechanism comes into play. We have made a quantitative assessment of how large the restricted volume can be and still lead to quantization of pH.
Poulsen
Kristian R
KR
Biostructure and Protein Engineering Group, Department of Physics and Nanotechnology, Aalborg University, Dk-9220 Aalborg, Denmark.
Snabe
Torben
T
Petersen
Evamaria I
EI
Fojan
Peter
P
Neves-Petersen
Maria T
MT
Wimmer
Reinhard
R
Petersen
Steffen B
SB
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D016956:Burkholderia cepacia / Q000201:enzymology; D005670:Fusarium / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008049:Lipase / Q000737:chemistry* / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D020103:Rhizomucor / Q000201:enzymology; D017550:Spectroscopy, Fourier Transform Infrared
D001426:Bacterial Proteins; D008049:Lipase