Cloned (Comment) | Organism |
---|---|
gene APN1, sequence comparisons, recombinant expression of His-tagged wild-type and mutant enzymes | Saccharomyces cerevisiae |
Protein Variants | Comment | Organism |
---|---|---|
H83A | site-directed mutagenesis, the mutation decrease the AP endonuclease activity of Apn1 owing to weak coordination of Zn2+ ions involved in enzymatic catalysis, suppressed enzymatic activity of H83A Apn1 results from the reduced number of active site Zn2+ ions. Analysis of kinetics of recognition, binding, and incision of DNA substrates of the H83A Apn1 mutant. Substitution of His83 with Ala influences catalytic complex formation and further incision of the damaged DNA strand. The H83A Apn1 catalysis depends not only on the location of the mismatch relative to the abasic site in DNA, but also on the nature of damage. H83A Apn1 appears to cleave substrates AP(2-aPu) and F(2-aPu) in several stages (F is tetrahydrofuran). Minimal kinetic mechanism of abasic site cleavage by H83A Apn1, molecular dynamics of H83A Apn1, overview. Molecular dynamics simulations of the H83A Apn1 structure containing the two Zn2+ ions reveal an insignificant movement of Zn2 relative to DNA and amino acid residues involved in Zn2 coordination. Structure of enzyme mutant H83A Apn1-substrate DNA complex with three Zn2+ ions containing Zn2+ ions per molecule of mutant enzyme, overview | Saccharomyces cerevisiae |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | measurement of conformational dynamics of DNA at pre-steady-state conditions, stopped-flow measurements. Rate and equilibrium constants for wild-type enzyme and mutant H83A Apn1 interactions with DNA substrates F(2-aPu) and AP(2-aPu), F is tetrahydrofuran | Saccharomyces cerevisiae |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Zn2+ | His83 coordinates one of three Zn2+ ions in Apn1's active site, structure comparisons. Structure of enzyme mutant H83A Apn1-substrate DNA complex with three Zn2+ ions containing Zn2+ ions per molecule of mutant enzyme, overview. Zn2+ ions are involved in catalysis | Saccharomyces cerevisiae |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Saccharomyces cerevisiae | P22936 | - |
- |
Saccharomyces cerevisiae ATCC 204508 / S288c | P22936 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes by nickel affinity chromatography | Saccharomyces cerevisiae |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | substrate oligodeoxyribonucleotides (ODNs) are synthesized and purified, a fluorescent 2-aminopurine (2-aPu) probe is located either on the 5'- or 3'-side of an abasic site. Cleavage of substrates AP(2-aPu) and F(2-aPu) in several stages (F is tetrahydrofuran) by wild-type and mutant H83A enzymes, overview | Saccharomyces cerevisiae | ? | - |
? | |
additional information | substrate oligodeoxyribonucleotides (ODNs) are synthesized and purified, a fluorescent 2-aminopurine (2-aPu) probe is located either on the 5'- or 3'-side of an abasic site. Cleavage of substrates AP(2-aPu) and F(2-aPu) in several stages (F is tetrahydrofuran) by wild-type and mutant H83A enzymes, overview | Saccharomyces cerevisiae ATCC 204508 / S288c | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
APN1 | - |
Saccharomyces cerevisiae |
apurinic/apyrimidinic endonuclease | - |
Saccharomyces cerevisiae |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Saccharomyces cerevisiae |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.6 | - |
assay at | Saccharomyces cerevisiae |
General Information | Comment | Organism |
---|---|---|
additional information | residue His83 properly coordinates the active site Zn2+ ion playing a crucial role in catalytic incision stage. Substrate binding structure analysis using DNA duplex crystal structure (PDB ID 2NQJ) for molecular dynamics simulations | Saccharomyces cerevisiae |
physiological function | the apurinic/apyrimidinic (AP) endonuclease Apn1 from Saccharomyces cerevisiae is a key enzyme involved in the base excision repair (BER) at the cleavage stage of abasic sites (AP sites) in DNA | Saccharomyces cerevisiae |