Probing the role of glutamic acid 144 in the EcoRI endonuclease using aspartic acid and glutamine replacements.
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.
Hager
P W
PW
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Reich
N O
NO
Day
J P
JP
Coche
T G
TG
Boyer
H W
HW
Rosenberg
J M
JM
Greene
P J
PJ
D001483:Base Sequence; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry; D015246:Deoxyribonuclease EcoRI / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology; D005971:Glutamates; D006860:Hydrogen Bonding; D007700:Kinetics; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D004268:DNA-Binding Proteins; D005971:Glutamates; D009841:Oligonucleotides; D015246:Deoxyribonuclease EcoRI