Engineering of the pH-dependence of thermolysin activity as examined by site-directed mutagenesis of Asn112 located at the active site of thermolysin.
Asn112 is located at the active site of thermolysin, 5-8 A from the catalytic Zn2+ and catalytic residues Glu143 and His231. When Asn112 was replaced with Ala, Asp, Glu, Lys, His, and Arg by site-directed mutagenesis, the mutant enzymes N112D and N112E, in which Asn112 is replaced with Asp and Glu, respectively, were secreted as an active form into Escherichia coli culture medium, while the other four were not. In the hydrolysis of a neutral substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide, the kcat/Km values of N112D and N112E exhibited bell-shaped pH-dependence, as did the wild-type thermolysin (WT). The acidic pKa of N112D was 5.7 +/- 0.1, higher by 0.4 +/- 0.2 units than that of WT, suggesting that the introduced negative charge suppressed the protonation of Glu143 or Zn2+-OH. In the hydrolysis of a negatively charged substrate, N-carbobenzoxy-l-Asp-l-Phe methyl ester (ZDFM), the pH-dependence of kcat/Km of the mutants decreased with increase in pH from 5.5 to 8.5, while that of WT was bell-shaped. This difference might be explained by the electrostatic repulsion between the introduced Asp/Glu and ZDFM, suggesting that introducing ionizing residues into the active site of thermolysin might be an effective means of modifying its pH-activity profile.
Kusano
Masayuki
M
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502.
Yasukawa
Kiyoshi
K
Hashida
Yasuhiko
Y
Inouye
Kuniyo
K
D019943:Amino Acid Substitution / Q000235:genetics; D001216:Asparagine / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002364:Caseins / Q000378:metabolism; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006632:Histamine / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013816:Thermodynamics; D013820:Thermolysin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D002364:Caseins; D050505:Mutant Proteins; D018698:Glutamic Acid; D001216:Asparagine; D006632:Histamine; D008666:Metalloendopeptidases; D013820:Thermolysin; D015032:Zinc