Exploring the nucleotide-binding site in proteins by affinity labeling and site-directed mutagenesis.

Abstract:

:Affinity labeling with nucleoside polyphosphopyridoxals, especially those with 3 or 4 phosphate groups, was effective for identifying the lysyl residue(s) located at or near the binding site for ATP, GTP, UDP-Glc, or ADP-Glc in various proteins. Furthermore, kinetic analysis of the mutant enzymes, in which the labeled lysyl residue was replaced with another amino acid by site-directed mutagenesis, provided evidence of its functional role. Affinity labeling of the mutant enzymes was useful for further identification of the hidden lysyl residue, which is unreactive in the wild-type enzyme but catalytically important. Comparison of the results of affinity labeling with different substrate analogues provided the information on the location of the labeled lysyl residue around the bound substrate. The affinity labeling reflected structural features of proteins, including their conformational flexibility.

journal_name

J Biochem

journal_title

Journal of biochemistry

authors

Fukui T

doi

10.1093/oxfordjournals.jbchem.a124834

subject

Has Abstract

pub_date

1995-06-01 00:00:00

pages

1139-44

issue

6

eissn

0021-924X

issn

1756-2651

journal_volume

117

pub_type

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