Alteration of the Specificity of Malate Dehydrogenase by Chemical Modulation of an Active Site Arginine
Malate dehydrogenase from Escherichia coli is highly specific for the oxidation of malate to oxaloacetate. The technique of site-specific modulation has been used to alter the substrate binding site of this enzyme. Introduction of a cysteine in place of the active site binding residue arginine 153 r...
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Published in | The Journal of biological chemistry Vol. 276; no. 33; pp. 31151 - 31155 |
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Main Authors | , |
Format | Journal Article |
Language | English |
Published |
United States
American Society for Biochemistry and Molecular Biology
17.08.2001
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Subjects | |
Online Access | Get full text |
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Summary: | Malate dehydrogenase from Escherichia coli is highly specific for the oxidation of malate to oxaloacetate. The technique of site-specific modulation has been used to
alter the substrate binding site of this enzyme. Introduction of a cysteine in place of the active site binding residue arginine
153 results in a mutant enzyme with diminished catalytic activity, but with K
m values for malate and oxaloacetate that are surprisingly unaffected. Reaction of this introduced cysteine with a series of
amino acid analog reagents leads to the incorporation of a range of functional groups at the active site of malate dehydrogenase.
The introduction of a positively charged group such as an amine or an amidine at this position results in improved affinity
for several inhibitors over that observed with the native enzyme. However, the recovery of catalytic activity is less dramatic,
with less than one third of the native activity achieved with the optimal reagents. These modified enzymes do have altered
substrate specificity, with α-ketoglutarate and hydroxypyruvate no longer functioning as alternative substrates. |
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Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 |
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M100892200 |