Structural Analyses of a Malate Dehydrogenase with a Variable Active Site
Malate dehydrogenase specifically oxidizes malate to oxaloacetate. The specificity arises from three arginines in the active site pocket that coordinate the carboxyl groups of the substrate and stabilize the newly forming hydroxyl/keto group during catalysis. Here, the role of Arg-153 in distinguish...
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Published in | The Journal of biological chemistry Vol. 276; no. 33; pp. 31156 - 31162 |
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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 specifically oxidizes malate to oxaloacetate. The specificity arises from three arginines in the active
site pocket that coordinate the carboxyl groups of the substrate and stabilize the newly forming hydroxyl/keto group during
catalysis. Here, the role of Arg-153 in distinguishing substrate specificity is examined by the mutant R153C. The x-ray structure
of the NAD binary complex at 2.1 Ã
reveals two sulfate ions bound in the closed form of the active site. The sulfate that
occupies the substrate binding site has been translated â¼2 Ã
toward the opening of the active site cavity. Its new location
suggests that the low catalytic turnover observed in the R153C mutant may be due to misalignment of the hydroxyl or ketone
group of the substrate with the appropriate catalytic residues. In the NAD·pyruvate ternary complex, the monocarboxylic inhibitor
is bound in the open conformation of the active site. The pyruvate is coordinated not by the active site arginines, but through
weak hydrogen bonds to the amide backbone. Energy minimized molecular models of unnatural analogues of R153C (Wright, S. K.,
and Viola, R. E. (2001) J. Biol. Chem. 276, 31151â31155) reveal that the regenerated amino and amido side chains can form favorable hydrogen-bonding interactions
with the substrate, although a return to native enzymatic activity is not observed. The low activity of the modified R153C
enzymes suggests that precise positioning of the guanidino side chain is essential for optimal orientation of the substrate. |
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Bibliography: | ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 |
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M100902200 |