Crystallization (Comment) | Organism |
---|---|
purified recombinant wild-type enzyme or mutants E48D, Y93F, D115E, D117E, D120E, D120N, and D152E in 60 mM MES, pH 6.0, and 10 mM Mg2+, 4°C, 0.008 ml sitting drops in the presence of 5 mM Mg2+, 1 mM PO43-, and a MPD concentration gradient from 16 to 19%, 2-4 weeks, cryoprotection by soaking of crystals at 4°C in 32% MPD, 30 mM MES, 10 mM Mg2+, and 1 mM PO43- for a few min, X-ray diffraction structure determination and analysis at 1.5-1.9 A resolution | Saccharomyces cerevisiae |
Protein Variants | Comment | Organism |
---|---|---|
D115E | site-directed mutagenesis, the mutation affects metal binding and the hydrogen bonding network in the active, in contrary to the wild-type enzyme, the mutant shows an open conformation variant of the hitherto unobserved two-phosphate and two bridging water active site, crystal structure determination with bound phosphate and Mg2+, and comparison to the wild-type enzyme structure | Saccharomyces cerevisiae |
D117E | site-directed mutagenesis, crystal structure determination with bound phosphate and Mg2+, and comparison to the wild-type enzyme structure | Saccharomyces cerevisiae |
D120E | site-directed mutagenesis, crystal structure determination with bound phosphate and Mg2+, and comparison to the wild-type enzyme structure | Saccharomyces cerevisiae |
D120N | site-directed mutagenesis, crystal structure determination with bound phosphate and Mg2+, and comparison to the wild-type enzyme structure | Saccharomyces cerevisiae |
D152E | site-directed mutagenesis, crystal structure determination with bound phosphate and Mg2+, and comparison to the wild-type enzyme structure | Saccharomyces cerevisiae |
E48D | site-directed mutagenesis, crystal structure determination with bound phosphate and Mg2+, and comparison to the wild-type enzyme structure | Saccharomyces cerevisiae |
Y93F | site-directed mutagenesis, the mutation affects metal binding and the hydrogen bonding network in the active, crystal structure determination with bound phosphate and Mg2+, and comparison to the wild-type enzyme structure | Saccharomyces cerevisiae |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | metal binding affinities and kinetics of wild-type and mutant enzymes, overview | Saccharomyces cerevisiae |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | the pyrophosphatase requires four divalent metal cations for catalysis, magnesium provides the highest activity | Saccharomyces cerevisiae |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
diphosphate + H2O | Saccharomyces cerevisiae | - |
2 phosphate | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Saccharomyces cerevisiae | P00817 | - |
- |
Reaction | Comment | Organism | Reaction ID |
---|---|---|---|
diphosphate + H2O = 2 phosphate | substrate binding structure and catalytic mechanism | Saccharomyces cerevisiae |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
diphosphate + H2O | - |
Saccharomyces cerevisiae | 2 phosphate | - |
r | |
diphosphate + H2O | the rate-determining step for the forward reaction with Mg2+ is hydrolysis of PPi, the wild-type active site shows a closed comformation with one of the two product phosphates already dissociated, active site residues Tyr93 and Asp115 are important, six-state catalytic mechanism, overview | Saccharomyces cerevisiae | 2 phosphate | in the reverse, net synthesis direction, the rate-determining step is not the condensation of the two phosphate ions but the previous step, which involves isomerization of the enzyme | r |
Synonyms | Comment | Organism |
---|---|---|
inorganic pyrophosphatase | - |
Saccharomyces cerevisiae |
PPase | - |
Saccharomyces cerevisiae |