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Membrane-bound pyrophosphatase of Thermotoga maritima requires sodium for activity
Tekijät: Belogurov GA, Malinen AM, Turkina MV, Jalonen U, Rytkonen K, Baykov AA, Lahti R
Kustantaja: AMER CHEMICAL SOC
Julkaisuvuosi: 2005
Journal: Biochemistry
Tietokannassa oleva lehden nimi: BIOCHEMISTRY
Lehden akronyymi: BIOCHEMISTRY-US
Vuosikerta: 44
Numero: 6
Aloitussivu: 2088
Lopetussivu: 2096
Sivujen määrä: 9
ISSN: 0006-2960
DOI: https://doi.org/10.1021/bi048429g
Tiivistelmä
Membrane-bound pyrophosphatase of the hyperthermophilic bacterium Thermotoga maritima (Tm-PPase), a homologue of H+-translocating pyrophosphatase, was expressed in Escherichia coli and isolated as inner membrane vesicles. In contrast to all previously studied H+-PPases, both native and recombinant Tm-PPases exhibited an absolute requirement for Na+ but displayed the highest activity in the presence of millimolar levels of both Na+ and K+. Detergent-solubilized recombinant Tm-PPase was thermostable and retained the monovalent cation requirements of the membrane-embedded enzyme. Steady-state kinetic analysis of pyrophosphate hydrolysis by the wild-type enzyme suggested that two Na+ binding sites and one K+ binding site are involved in enzyme activation. The affinity of the site that binds Na+ first is increased with increasing K+ concentration. In contrast, only one Na+ binding site (K+-dependent) and one K+ binding site were involved in activation of the Asp(703) --> Asn variant. Thus, Asp(703) may form part of the K+-independent Na+ binding site. Unlike all other membrane and soluble PPases, Tm-PPase did not catalyze oxygen exchange between phosphate and water. However, solubilized Tm-PPase exhibited low but measurable PPi-synthesizing activity, which also required Na+ but was inhibited by K+. These results demonstrate that T. maritima PPase belongs to a previously unknown subfamily of Na+-dependent H+-PPase homologues and may be an analogue of Na+,K+-ATPase.
Membrane-bound pyrophosphatase of the hyperthermophilic bacterium Thermotoga maritima (Tm-PPase), a homologue of H+-translocating pyrophosphatase, was expressed in Escherichia coli and isolated as inner membrane vesicles. In contrast to all previously studied H+-PPases, both native and recombinant Tm-PPases exhibited an absolute requirement for Na+ but displayed the highest activity in the presence of millimolar levels of both Na+ and K+. Detergent-solubilized recombinant Tm-PPase was thermostable and retained the monovalent cation requirements of the membrane-embedded enzyme. Steady-state kinetic analysis of pyrophosphate hydrolysis by the wild-type enzyme suggested that two Na+ binding sites and one K+ binding site are involved in enzyme activation. The affinity of the site that binds Na+ first is increased with increasing K+ concentration. In contrast, only one Na+ binding site (K+-dependent) and one K+ binding site were involved in activation of the Asp(703) --> Asn variant. Thus, Asp(703) may form part of the K+-independent Na+ binding site. Unlike all other membrane and soluble PPases, Tm-PPase did not catalyze oxygen exchange between phosphate and water. However, solubilized Tm-PPase exhibited low but measurable PPi-synthesizing activity, which also required Na+ but was inhibited by K+. These results demonstrate that T. maritima PPase belongs to a previously unknown subfamily of Na+-dependent H+-PPase homologues and may be an analogue of Na+,K+-ATPase.
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