Characterization of a Monomeric Escherichia coli Alkaline Phosphatase Formed upon a Single Amino Acid Substitution
Alkaline phosphatase (AP) from Escherichia coli as well as APs from many other organisms exist in a dimeric quaternary structure. Each monomer contains an active site located 32 Ã away from the active site in the second subunit. Indirect evidence has previously suggested that the monomeric form of...
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Published in | The Journal of biological chemistry Vol. 278; no. 26; pp. 23497 - 23501 |
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Main Authors | , |
Format | Journal Article |
Language | English |
Published |
United States
American Society for Biochemistry and Molecular Biology
27.06.2003
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Subjects | |
Online Access | Get full text |
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Summary: | Alkaline phosphatase (AP) from Escherichia coli as well as APs from many other organisms exist in a dimeric quaternary structure. Each monomer contains an active site
located 32 Ã
away from the active site in the second subunit. Indirect evidence has previously suggested that the monomeric
form of AP is inactive. Molecular modeling studies indicated that destabilization of the dimeric interface should occur if
Thr-59, located near the 2-fold axis of symmetry, were replaced by a sterically large and charged residue such as arginine.
The T59R enzyme was constructed and characterized by sucrose-density gradient sedimentation, size-exclusion chromatography,
and circular dichroism (CD) and compared with the previously constructed T59A enzyme. The T59A enzyme was found to exist
as a dimer, whereas the T59R enzyme was found to exist as a monomer. The T59A, T59R, and wild-type APs exhibited almost
identical secondary structures as judged by CD. The T59R monomeric AP has a melting temperature ( T m ) of 43 °C, whereas the wild-type AP dimer has a T m of 97 °C. The catalytic activity of the T59R enzyme was reduced by 10 4 -fold, whereas the T59A enzyme exhibited an activity similar to that of the wild-type enzyme. The T59A and wild-type enzymes
contained similar levels of zinc and magnesium, whereas the T59R enzyme has almost undetectable amounts of tightly bound
metals. These results suggest that a significant conformational change occurs upon dimerization, which enhances thermal
stability, metal binding, and catalysis. |
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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.M301105200 |