An Examination by Site-Directed Mutagenesis of Putative Key Residues in the Determination of Coenzyme Specificity in Clostridial NAD+-Dependent Glutamate Dehydrogenase
Joint Authors
Source
Issue
Vol. 2011, Issue 2011 (31 Dec. 2011), pp.1-9, 9 p.
Publisher
Hindawi Publishing Corporation
Publication Date
2011-08-16
Country of Publication
Egypt
No. of Pages
9
Main Subjects
Abstract EN
Sequence and structure comparisons of various glutamate dehydrogenases (GDH) and other nicotinamide nucleotide-dependent dehydrogenases have potentially implicated certain residues in coenzyme binding and discrimination.
We have mutated key residues in Clostridium symbiosum NAD+-specific GDH to investigate their contribution to specificity and to enhance acceptance of NADPH.
Comparisons with E.
coli NADPH-dependent GDH prompted design of mutants F238S, P262S, and F238S/P262S, which were purified and assessed at pH 6.0, 7.0, and 8.0.
They showed markedly increased catalytic efficiency with NADPH, especially at pH 8.0 (∼170-fold for P262S and F238S/P262S with relatively small changes for NADH).
A positive charge introduced through the D263K mutation also greatly increased catalytic efficiency with NADPH (over 100-fold at pH 8) and slightly decreased activity with NADH.
At position 242, “P6” of the “core fingerprint,” where NAD+- and NADP+-dependent enzymes normally have Gly or Ala, respectively, clostridial GDH already has Ala.
Replacement with Gly produced negligible shift in coenzyme specificity.
American Psychological Association (APA)
Griffin, Joanna& Engel, Paul C.. 2011. An Examination by Site-Directed Mutagenesis of Putative Key Residues in the Determination of Coenzyme Specificity in Clostridial NAD+-Dependent Glutamate Dehydrogenase. Enzyme Research،Vol. 2011, no. 2011, pp.1-9.
https://search.emarefa.net/detail/BIM-483741
Modern Language Association (MLA)
Griffin, Joanna& Engel, Paul C.. An Examination by Site-Directed Mutagenesis of Putative Key Residues in the Determination of Coenzyme Specificity in Clostridial NAD+-Dependent Glutamate Dehydrogenase. Enzyme Research No. 2011 (2011), pp.1-9.
https://search.emarefa.net/detail/BIM-483741
American Medical Association (AMA)
Griffin, Joanna& Engel, Paul C.. An Examination by Site-Directed Mutagenesis of Putative Key Residues in the Determination of Coenzyme Specificity in Clostridial NAD+-Dependent Glutamate Dehydrogenase. Enzyme Research. 2011. Vol. 2011, no. 2011, pp.1-9.
https://search.emarefa.net/detail/BIM-483741
Data Type
Journal Articles
Language
English
Notes
Includes bibliographical references
Record ID
BIM-483741