Single-point amino acid substitutions at the 119th residue of thermolysin and their pressure-induced activation.
The effect of amino acid substitution at the 119th site of thermolysin (TLN) on the pressure activation behavior of this enzyme was studied for four mutants at pressures < 300 MPa. For Q119Q, Q119N and Q119R, the highest activation was observed to be over 30 times that at atmospheric pressure and the activation volumes (deltaV++) were about -75 ml/mol. However, we obtained only 10 times higher activation for Q119E and Q119D (deltaV++ approximately -60 ml/mol). The intrinsic fluorescence of TLN changed at pressures > 300 MPa, and the latter two mutants showed a smaller deltaGapp and deltaVapp of transition than the wild type. These results are discussed with respect to the hydration change in the enzyme protein around the substituted region.
Kunugi
S
S
Department of Polymer Science and Engineering, Kyoto Institute of Technology, Sakyo, Japan. kunugi@ipc.kit.ac.jp
Fujiwara
S
S
Kidokoro
S
S
Endo
K
K
Hanzawa
S
S
D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry*; D006874:Hydrostatic Pressure; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013820:Thermolysin / Q000737:chemistry* / Q000378:metabolism*
D000596:Amino Acids; D013820:Thermolysin