A1 Vertaisarvioitu alkuperäisartikkeli tieteellisessä lehdessä
Structural and functional consequences of substitutions at the tyrosine 55 lysine 104 hydrogen bond in Escherichia coli inorganic pyrophosphatase
Tekijät: Fabrichniy IP, Kasho VN, Hyytia T, Salminen T, Halonen P, Dudarenkov VY, Heikinheimo P, Chernyak VY, Goldman A, Lahti R, Cooperman BS, Baykov AA
Kustantaja: AMER CHEMICAL SOC
Julkaisuvuosi: 1997
Tietokannassa oleva lehden nimi: BIOCHEMISTRY
Lehden akronyymi: BIOCHEMISTRY-US
Vuosikerta: 36
Numero: 25
Aloitussivu: 7746
Lopetussivu: 7753
Sivujen määrä: 8
ISSN: 0006-2960
DOI: https://doi.org/10.1021/bi9629844
Tiivistelmä
Tyrosine 55 and lysine 104 are evolutionarily conserved residues that form a hydrogen bond in the active site of Escherichia coli inorganic pyrophosphatase (E-PPase). Here we used site-directed mutagenesis to examine their roles in structure stabilization and catalysis. Though these residues are not part of the subunit interface, Y55F and K104R (but not K104I) substitutions markedly destabilize the hexameric structure, allowing dissociation into active trimers on dilution. A K104I variant is nearly inactive while Y55F and K104R variants exhibit appreciable activity and require greater concentrations of Mg2+ and higher pH for maximal activity. The effects on activity are explained by (a) increased pK(a)s for the catalytically essential base and acid at the active site, (b) decreases in the rate constant for substrate (dimagnesium pyrophosphate) binding to enzyme-Mg-2 complex vs enzyme-Mg-3 complex, and (c) parallel decreases in the catalytic constant for the resulting enzyme-Mg-2-substrate and enzyme-Mg-3-substrate complexes. The results are consistent with the major structural roles of Tyr55 and Lys104 in the active site. The microscopic rate constant for PPi hydrolysis on either the Y55F or K104R variants increases, by a factor of 3-4 in the pH range 7.2-8.0, supporting the hypothesis that this reaction step depends on an essential base within the enzyme active site.
Tyrosine 55 and lysine 104 are evolutionarily conserved residues that form a hydrogen bond in the active site of Escherichia coli inorganic pyrophosphatase (E-PPase). Here we used site-directed mutagenesis to examine their roles in structure stabilization and catalysis. Though these residues are not part of the subunit interface, Y55F and K104R (but not K104I) substitutions markedly destabilize the hexameric structure, allowing dissociation into active trimers on dilution. A K104I variant is nearly inactive while Y55F and K104R variants exhibit appreciable activity and require greater concentrations of Mg2+ and higher pH for maximal activity. The effects on activity are explained by (a) increased pK(a)s for the catalytically essential base and acid at the active site, (b) decreases in the rate constant for substrate (dimagnesium pyrophosphate) binding to enzyme-Mg-2 complex vs enzyme-Mg-3 complex, and (c) parallel decreases in the catalytic constant for the resulting enzyme-Mg-2-substrate and enzyme-Mg-3-substrate complexes. The results are consistent with the major structural roles of Tyr55 and Lys104 in the active site. The microscopic rate constant for PPi hydrolysis on either the Y55F or K104R variants increases, by a factor of 3-4 in the pH range 7.2-8.0, supporting the hypothesis that this reaction step depends on an essential base within the enzyme active site.
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