J. Biol. Chem., Vol. 265, Issue 35, 21520-21526, Dec, 1990
Probing the role of glutamic acid 144 in the EcoRI endonuclease using aspartic acid and glutamine replacements
PW Hager, NO Reich, JP Day, TG Coche, HW Boyer, JM Rosenberg and PJ Greene
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
The x-ray structure of the EcoRI endonuclease-DNA complex (3) suggests that
hydrogen bonds between amino acids, glutamic acid 144, arginine 145, and
arginine 200, and major groove base moieties are the molecular determinants
of specificity. We have investigated residue 144 using aspartate and
glutamine substitutions introduced by site-directed mutagenesis.
Substitution with glutamine results in a null phenotype (at least a
2000-fold reduction in activity). On the other hand, the aspartic acid
mutant (ED144) retained in vivo activity. Substrate binding and catalytic
studies were done with purified ED144 enzyme. The affinity of the ED144
enzyme for the canonical sequence 5'-GAATTC-3' is about 340-fold less than
the wild-type (WT) enzyme, while its affinity for nonspecific DNA is about
50 times greater. The ED144 enzyme cleaves one strand in the EcoRI site in
plasmid pBR322 with a kcat/Km similar to WT. In contrast to the WT enzyme,
the ED144 enzyme dissociates after the first strand cleavage. Partitioning
between cleavage and dissociation at the first and second cleavage steps
for the ED144 enzyme is extremely salt-sensitive. The altered partitioning
results largely from a destabilization of the enzyme-DNA complex,
particularly the enzyme-nicked DNA complex, with only small changes in the
respective cleavage rates. The hydrogen bonds of Glu-144 are critical, they
appear to act cooperatively with other specificity contacts to stabilize
the enzyme-DNA complex.
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