Characterization of three types of aspartase activated by site-directed mutagenesis, limited proteolysis, and acetylation.
The activity of aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) from Escherichia coli is enhanced 2- to 3-fold by three types of modification of the enzyme as reported previously; the replacement of Cys-430 with Trp by site-directed mutagenesis, the truncation of the C-terminal region by limited proteolysis, and the acetylation of amino groups with acetic anhydride. To elucidate the molecular basis of such activation, we have compared the kinetic properties of the modified enzymes in this study. Although the modifications caused very similar changes in the kinetic properties, such as increase in kcat, the half-saturating concentration of substrate, and Hill coefficient values, the modified enzymes differed greatly in sensitivity to the activator L-aspartate and the inhibitor Cl- ions. As a result of the mutation, the binding affinity for the activator was greatly decreased without change in the sensitivity to the inhibitor, whereas after acetylation, the sensitivity to the inhibitor was completely lost without decrease in the binding affinity for the activator. After truncation of the C-terminal region, both a large decrease in the binding affinity for the activator and complete loss of sensitivity to the inhibitor occurred, suggesting that this type of activation is equivalent to the former two types combined.
Murase
S
S
Department of Chemistry, Faculty of Science, Kyoto University.
Yumoto
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N
D000089:Acetic Anhydrides; D000107:Acetylation; D001220:Aspartate Ammonia-Lyase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D003545:Cysteine; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin / Q000378:metabolism
D000089:Acetic Anhydrides; D002712:Chlorides; C031800:acetic anhydride; D001224:Aspartic Acid; D014357:Trypsin; D001220:Aspartate Ammonia-Lyase; D003545:Cysteine