Kinetic analysis of enhanced thermal stability of an alkaline protease with engineered twin disulfide bridges and calcium-dependent stability.
The thermal stability of a cysteine-free alkaline protease (Alp) secreted by the eukaryote Aspergillus oryzae was improved both by the introduction of engineered twin disulfide bridges (Cys-69/Cys-101 and Cys-169/Cys-200), newly constructed as part of this study, and by the addition of calcium ions. We performed an extensive kinetic analysis of the increased thermal stability of the mutants as well as the role of calcium dependence. The thermodynamic activation parameters for irreversible thermal inactivation, the activation free energy (deltaG), the activation enthalpy (deltaH), and the activation entropy (deltaS) were determined from absolute reaction rate theory. The values of deltaH and deltaS were significantly and concomitantly increased as a result of introducing the twin disulfide bridges, for which the increase in the value of deltaH outweighed that of deltaS, resulting in significant increases in the value of deltaG. The enhancement of the thermal stability obtained by introducing the twin disulfide bridges is an example of the so-called low-temperature stabilization of enzymes. The stabilizing effect of calcium ions on wild-type Alp is similar to the results we obtained by introducing the engineered twin disulfide bridges.
Ikegaya
Kazuo
K
Pharmaceutical Research Division, Mitsubishi Pharma Corporation, 2-25-1 Shodai-Ohtani, Hirakata, Osaka 573-1153, Japan. Ikegaya.Kazuo@mk.m-pharma.co.jp
Sugio
Shigetoshi
S
Murakami
Kohji
K
Yamanouchi
Kouichi
K
D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000378:metabolism*; D004795:Enzyme Stability; D005818:Genetic Engineering; D006207:Half-Life; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics
D004220:Disulfides; D012697:Serine Endopeptidases; C032923:microbial serine proteinases; D003545:Cysteine; D002118:Calcium