Effects of Site-directed Mutagenesis of the Surface Residues Gln128 and Gln225 of Thermolysin on its Catalytic Activity
| Autor: | Chika Tatsumi, Kiyoshi Yasukawa, Kuniyo Inouye, Yasuhiko Hashida |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
Surface Properties
Stereochemistry Glutamine DNA Mutational Analysis Molecular Conformation Thermolysin Bacillus Stereoisomerism Biochemistry Catalysis Binding site Site-directed mutagenesis Molecular Biology chemistry.chemical_classification Binding Sites biology Chemistry Mutagenesis Temperature Active site Substrate (chemistry) General Medicine Hydrogen-Ion Concentration Kinetics Zinc Enzyme Metalloproteases Mutagenesis Site-Directed biology.protein |
| Zdroj: | Journal of Biochemistry. 141:835-842 |
| ISSN: | 0021-924X |
| DOI: | 10.1093/jb/mvm087 |
| Popis: | Thermolysin is remarkably activated and stabilized by neutral salts with varying degrees depending on salt species, and particular surface residues are thought to be especially important in its activity and stability [Inouye, K. (1992) J. Biochem. 112, 335-340; Inouye, K. et al. (1998) Biochim. Biophys. Acta 1388, 209-214]. In this study, we examined the mutational effects of the surface residues of thermolysin. Gln128 and Gln225 were selected as the residues to be mutated because they are located on the surface loop and close to but not in the active site (23.5 and 15.8 A far from the active site zinc ion, respectively) and fully solvent accessible. Nine single mutants [Q128K (Gln128 is replaced with Lys), Q128E, Q128A, Q225K, Q225R, Q225E, Q225D, Q225A and Q225V] were constructed by site-directed mutagenesis. Mutational changes in catalytic activity were found only in the mutant thermolysins having a hydrophobic residue at the position 225 (Q225A and Q225V). In the hydrolysis of a neutral substrate N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide (FAGLA), the alkaline pK(a) value of Q225A is 8.48 +/- 0.04, being higher by 0.42 +/- 0.07 units than that of the wild-type thermolysin. The k(cat)/K(m) value of the wild-type enzyme is enhanced 14 times with 4 M NaCl, and those of Q225A and Q225V are enhanced 10 and 19 times, respectively. In the hydrolysis of a negatively charged substrate N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester (ZDFM), unlike FAGLA, the initial velocities of Q225A and Q225V decreased to 30 and 50% of that of the wild-type enzyme, respectively. Their thermal stability is similar to that of the wild-type enzyme. These findings indicate that even a single mutation at the thermolysin surface induces changes in the electrostatic environment in the active site and affects the activity. Thus, site-directed mutagenesis of surface residues of thermolysin, including apparently thermodynamically unfavorable introduction of hydrophobic residues, should be explored to improve its activity and stability. |
| Databáze: | OpenAIRE |
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