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The Binding of Oxidised Coenzyme to Bovine-Liver Glutamate Dehydrogenase Studied by Circular-Difference Spectroscopy
| Content Provider | Scilit |
|---|---|
| Author | O'Neill, Kevin T. J. Bayley, Peter M. |
| Copyright Year | 2005 |
| Description | Journal: Journal of Biological Inorganic Chemistry 1. The binding of NAD+ to glutamate dehydrogenase may be followed quantitatively by titration, using high-sensitivity circular dichroism (CD) difference spectroscopy. 2. The CD of the bound coenzyme in the binary complex E . NAD closely resembles that of bound ADP, although the affinity is much lower, being 350-fold less for NAD+ at 20 degrees C in 0.1 M phosphate, pH 7. 3. A family of CD spectra may be analysed by unconstrained linear regression assuming only three components: free enzyme, free coenzyme, and a single binary complex, E . NAD. 4. Taking the molar CD of bound ADP as representing the molar CD of the adenine chromophore of bound NAD+, the linear regression shows the formation of a simple 1 : 1 complex E . NAD with Kd = 0.72 mM in a simple binding process without positive or negative cooperativity. 5. NADP+ binding is more than 10-fold weaker than NAD+ binding. 6. From the similarity of the CD of bound ADP and bound NAD+ it is probable that NAD+, in forming a simple binary complex, binds preferentially at the regulatory (adenine nucleotide) binding site (site II). 7. Direct evidence has been obtained for the binding of a second molecule of NAD+ to the ternary complex E . NAD . glutarate. This process occurs with low affinity and is probably also located at the adenine regulatory site. 8. This second-site binding of NAD+ may contribute to the phenomena of non-Michaelis-Menten kinetics and apparent negative homotropic interactions in the binding of NAD+, previously attributed to subunit-subunit cooperative interactions. |
| Related Links | http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1980.tb06115.x/pdf |
| Ending Page | 531 |
| Page Count | 11 |
| Starting Page | 521 |
| ISSN | 09498257 |
| e-ISSN | 14321327 |
| DOI | 10.1111/j.1432-1033.1980.tb06115.x |
| Journal | Journal of Biological Inorganic Chemistry |
| Issue Number | 3 |
| Volume Number | 112 |
| Language | English |
| Publisher | Wiley-Blackwell |
| Publisher Date | 2005-03-03 |
| Access Restriction | Open |
| Subject Keyword | Journal: Journal of Biological Inorganic Chemistry Biochemistry and Molecular Biology Bound Adp |
| Content Type | Text |
| Resource Type | Article |
| Subject | Biochemistry Inorganic Chemistry |
