Pressure enhances thermal stability of DNA polymerase from three thermophilic organisms.
DNA polymerases derived from three thermophilic microorganisms, Pyrococcus strain ES4, Pyrococcus furiosus, and Thermus aquaticus, were stabilized in vitro by hydrostatic pressure at denaturing temperatures of 111 degrees C, 107.5 degrees C, and 100 degrees C (respectively). Inactivation rates, as determined by enzyme activity measurements, were measured at 3, 45, and 89 MPa. Half-lives of P. strain ES4, P. furiosus, and T. aquaticus DNA polymerases increased from 5.0, 6.9, and 5.2 minutes (respectively) at 3 MPa to 12, 36, and 13 minutes (respectively) at 45 MPa. A pressure of 89 MPa further increased the half-lives of P. strain ES4 and T. aquaticus DNA polymerases to 26 and 39 minutes, while the half-life of P. furiosus DNA polymerase did not increase significantly from that at 45 MPa. The decay constant for P. strain ES4 and T. aquaticus polymerases decreased exponentially with increasing pressure, reflecting an observed change in volume for enzyme inactivation of 61 and 73 cm3/mol, respectively. Stabilization by pressure may result from pressure effects on thermal unfolding or pressure retardation of unimolecular inactivation of the unfolded state. Regardless of the mechanism, pressure stabilization of proteins could explain the previously observed extension of the maximum temperature for survival of P. strain ES4 and increase the survival of thermophiles in thermally variable deep-sea environments such as hydrothermal vents.
Summit
M
M
School of Oceanography, University of Washington, Seattle 98195, USA. summit@ocean.washington.edu
Scott
B
B
Nielson
K
K
Mathur
E
E
Baross
J
J
D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D004777:Environment; D004795:Enzyme Stability; D006207:Half-Life; D006358:Hot Temperature; D006874:Hydrostatic Pressure; D007700:Kinetics; D019384:Nucleic Acid Synthesis Inhibitors; D011489:Protein Denaturation; D019711:Pyrococcus / Q000201:enzymology*; D020027:Pyrococcus furiosus / Q000201:enzymology; D012623:Seawater / Q000382:microbiology; D013824:Thermus / Q000201:enzymology*
D019384:Nucleic Acid Synthesis Inhibitors; D004259:DNA-Directed DNA Polymerase