English
 
User Manual Privacy Policy Disclaimer Contact us
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT
  Fluorescence and NMR investigations on the ligand binding properties of adenylate kinases

Reinstein, J., Vetter, I. R., Schlichting, I., Rösch, P., Wittinghofer, A., & Goody, R. S. (1990). Fluorescence and NMR investigations on the ligand binding properties of adenylate kinases. Biochemistry, 29(32), 7440-7450. doi:10.1021/bi00484a013.

Item is

Basic

show hide
Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0019-ACE9-4 Version Permalink: http://hdl.handle.net/21.11116/0000-0002-6A01-6
Genre: Journal Article

Files

show Files
hide Files
:
Biochem_29_1990_7440.pdf (Any fulltext), 2MB
 
File Permalink:
-
Name:
Biochem_29_1990_7440.pdf
Description:
-
Visibility:
Restricted (Max Planck Institute for Medical Research, MHMF; )
MIME-Type / Checksum:
application/pdf
Technical Metadata:
Copyright Date:
-
Copyright Info:
-
License:
-

Locators

show
hide
Description:
-
Locator:
https://dx.doi.org/10.1021/bi00484a013 (Any fulltext)
Description:
-

Creators

show
hide
 Creators:
Reinstein, Jochen1, Author              
Vetter, Ingrid R., Author
Schlichting, Ilme2, Author              
Rösch, Paul2, Author              
Wittinghofer, Alfred2, Author              
Goody, Roger S.2, Author              
Affiliations:
1Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society, ou_1497700              
2Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society, ou_1497712              

Content

show
hide
Free keywords: -
 Abstract: A new system for measurement of affinities of adenylate kinases (AK) for substrates and inhibitors is presented. This system is based on the use of the fluorescent ligand alpha,omega-di[(3' or 2')-O-(N-methylanthraniloyl)adenosine-5'] pentaphosphate (mAP5Am), which is an analogue of the bisubstrate inhibitor diadenosine pentaphosphate (AP5A). It allows the determination of dissociation constants for any ligand in the range of 1 x 10(-9) to 5 x 10(-2) M. Affinities for different bisubstrate inhibitors (AP4A, AP5A, AP6A) and substrates (AMP, ADP, ATP, GTP) were determined in the presence and absence of magnesium. An analysis of the binding of bisubstrate inhibitors is proposed and applied to these data. The techniques are used to describe the properties of a mutant enzyme with Gln-28----His (Q28H) prepared by site-directed mutagenesis in comparison to those of wild-type AK from Escherichia coli. This newly introduced histidine is already present in most other adenylate kinases and was regarded to be important or even essential for the catalytic reaction of AK. Temperature denaturation experiments indicate that the mutant enzyme has the same thermal stability as the wild-type enzyme and, as NMR studies indicate, also a very similar structure. However, steady-state catalytic studies and binding experiments showed that the affinities for substrates and inhibitors are elevated from 3-fold (AMP) to 5-fold (ATP) to 15-fold (AP5A) compared to those of the wild-type enzyme. Together with the results obtained by Tian et al. [Tian, G., Sanders, C. R., Kishi, F., Nakazawa, A., & Tsai, M.-D. (1988) Biochemistry 27, 5544-5552] on the effect of replacement of the conserved His-36 in the cytosolic AK (AK1) from chicken by glutamine and asparagine, this shows that residues 28 of AK from E. coli (AKec) and 36 of AK1 are situated in a comparable environment and are not essential for catalytic activity.

Details

show
hide
Language(s): eng - English
 Dates: 1989-08-161990-04-021990-08-14
 Publication Status: Published in print
 Pages: 11
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Degree: -

Event

show

Legal Case

show

Project information

show

Source 1

show
hide
Title: Biochemistry
Source Genre: Journal
 Creator(s):
Affiliations:
Publ. Info: Columbus, Ohio : American Chemical Society
Pages: - Volume / Issue: 29 (32) Sequence Number: - Start / End Page: 7440 - 7450 Identifier: ISSN: 0006-2960
CoNE: https://pure.mpg.de/cone/journals/resource/954925384103