The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.

Derbyshire V, Grindley ND, Joyce CM
The EMBO journal (1991), Volume 10, Page 17
PubMed entry Full article text

Abstract:

We have used site-directed mutagenesis to change amino acid side ...
We have used site-directed mutagenesis to change amino acid side chains that have been shown crystallographically to be in close proximity to a DNA 3' terminus bound at the 3'-5' exonuclease active site of Klenow fragment. Exonuclease assays of the resulting mutant proteins indicate that the largest effects on exonuclease activity result from mutations in a group of carboxylate side chains (Asp355, Asp424 and Asp501) anchoring two divalent metal ions that are essential for exonuclease activity. Another carboxylate (Glu357) within this cluster seems to be less important as a metal ligand, but may play a separate role in catalysis of the exonuclease reaction. A second group of residues (Leu361, Phe473 and Tyr497), located around the terminal base and ribose positions, plays a secondary role, ensuring correct positioning of the substrate in the active site and perhaps also facilitating melting of a duplex DNA substrate by interacting with the frayed 3' terminus. The pH-dependence of the 3'-5' exonuclease reaction is consistent with a mechanism in which nucleophilic attack on the terminal phosphodiester bond is initiated by a hydroxide ion coordinated to one of the enzyme-bound metal ions.

Polymerases:

Eco Pol I,Klenow fragment,KF D355A,KF L361A,KF L361M,KF D424A,KF D424E,KF D424N

Topics:

Mutational Analysis, Historical Protein Properties (MW, pI, ...), Kinetic Parameters, Exonuclease Activity

One line summary:

Structure-function analysis using a panel of 3'-5'exo active site mutations.

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Klenow fragment The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. 3-5' Exonuclease (proofreading) Yes
Klenow fragment The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. Cloned or native Cloned in E. coli
Klenow fragment The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. Tagged No
Klenow fragment The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. Full length or truncated Full length
Klenow fragment The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. Specific Activity 9000 units/mg Technique: Polymerase assay (poly[d(AT)]
Klenow fragment The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. KM 0.56uM Reaction: 3-5' Exonuclease; Technique: Steady State (ssDNA: poly(dT)); Experimental conditions: pH (pH 7.5), Mg++ (6mM), Temp (37°C)
Klenow fragment The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. kcat 0.09 /second Reaction: 3-5' Exonuclease; Technique: Steady State (ssDNA: poly(dT)); Experimental conditions: pH (pH 7.5), Mg++ (6mM), Temp (37°C)
Klenow fragment The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. kcat 0.12 /second Reaction: 3-5' Exonuclease; Technique: Steady State (ssDNA: poly(dA)); Experimental conditions: pH (pH 7.5), Mg++ (8mM), Temp (37°C)
KF L361A The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. Specific Activity 9000 units/mg Technique: Polymerase assay (poly[d(AT)]
KF L361A The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. kcat 0.044 /second Reaction: 3-5' Exonuclease; Technique: Steady State (ssDNA, poly(dA)); Experimental conditions: pH (pH 7.5), Mg++ (8mM), Temp (37°C)
KF L361A The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. kcat 4E-05 /second Reaction: 3-5' Exonuclease; Technique: Steady State (dsDNA, assuming WT has rate 0.001/s); Experimental conditions: pH (pH 7.5), Mg++ (6mM), Temp (37°C)
KF L361M The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. Specific Activity 1.1E+04 units/mg Technique: Polymerase assay (poly[d(AT)]
KF L361M The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. kcat 8.3E-05 /second Reaction: 3-5' Exonuclease; Technique: Steady State (dsDNA, assuming WT has rate 0.001/s); Experimental conditions: pH (pH 7.5), Mg++ (6mM), Temp (37°C)
KF D424A The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. Specific Activity 9000 units/mg Technique: Polymerase assay (poly[d(AT)]
KF D424A The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. kcat 1.3E-08 /second Reaction: 3-5' Exonuclease; Technique: Steady State (dsDNA, assuming WT has rate 0.001/s); Experimental conditions: pH (pH 7.5), Temp (37°C)
KF D424E The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. Specific Activity 1.1E+04 units/mg Technique: Polymerase assay (poly[d(AT)]
KF D424E The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. kcat 0.01 /second Reaction: 3-5' Exonuclease; Technique: Steady State (ssDNA, poly(dA)); Experimental conditions: pH (pH 7.5), Mg++ (8mM), Temp (37°C)
KF D424E The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. kcat 4E-05 /second Reaction: 3-5' Exonuclease; Technique: Steady State (dsDNA, assuming WT has rate 0.001/s); Experimental conditions: pH (pH 7.5), Mg++ (6mM), Temp (37°C)
KF D355A The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. Specific Activity 1.3E+04 units/mg Technique: Polymerase assay (poly[d(AT)]
KF D355A The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. kcat 8.3E-08 /second Reaction: 3-5' Exonuclease; Technique: Steady State (dsDNA substrate, assuming WT has rate 0.001/s); Experimental conditions: pH (pH 7.5), Mg++ (6mM), Temp (37°C)
KF D424N The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. Specific Activity 1.2E+04 units/mg Technique: Polymerase assay (poly[d(AT)]
KF D424N The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction. kcat 2.5E-08 /second Reaction: 3-5' Exonuclease; Technique: Steady State (dsDNA, assuming WT has rate 0.001/s); Experimental conditions: pH (pH 7.5), Mg++ (6mM), Temp (37°C)
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