1b95
From Proteopedia
|
ANALYSIS OF A MUTATIONAL HOT-SPOT IN THE ECORV RESTRICTION ENDONUCLEASE: A CATALYTIC ROLE FOR A MAIN CHAIN CARBONYL GROUP
Overview
Following random mutagenesis of the Eco RV endonuclease, a high proportion, of the null mutants carry substitutions at Gln69. Such mutants display, reduced rates for the DNA cleavage step in the reaction pathway, yet the, crystal structures of wild-type Eco RV fail to explain why Gln69 is, crucial for activity. In this study, crystal structures were determined, for two mutants of Eco RV, with Leu or Glu at residue 69, bound to, specific DNA. The structures of the mutants are similar to the native, protein and no function can be ascribed to the side chain of the amino, acid at this locus. Instead, the structures of the mutant proteins suggest, that the catalytic defect is due to the positioning of the main chain, carbonyl group. In the enzyme-substrate complex for Eco RV, the main chain, carbonyl of Gln69 makes no interactions with catalytic functions but, in, the enzyme-product complex, it coordinates a metal ion bound to the newly, liberated 5'-phosphate. This re-positioning may be hindered in the mutant, proteins. Molecular dynamics calculations indicate that the metal on the, phosphoryl oxygen interacts with the carbonyl group upon forming the, pentavalent intermediate during phosphodiester hydrolysis. A main chain, carbonyl may thus play a role in catalysis by Eco RV.
About this Structure
1B95 is a Single protein structure of sequence from Escherichia coli. Active as Type II site-specific deoxyribonuclease, with EC number 3.1.21.4 Full crystallographic information is available from OCA.
Reference
Structural analysis of a mutational hot-spot in the EcoRV restriction endonuclease: a catalytic role for a main chain carbonyl group., Thomas MP, Brady RL, Halford SE, Sessions RB, Baldwin GS, Nucleic Acids Res. 1999 Sep 1;27(17):3438-45. PMID:10446231
Page seeded by OCA on Tue Nov 20 11:28:20 2007
