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Nucleotide- and substrate-induced conformational transitions in the CBS domain-containing pyrophosphatase of Moorella thermoacetica.

Abstract：

:In contrast to all other known pyrophosphatases, Moorella thermoacetica pyrophosphatase (mtCBS-PPase) contains nucleotide-binding CBS domains and is thus strongly regulated by adenine nucleotides. Stopped-flow measurements using a fluorescent AMP analogue, 2'(3')-O-(N-methylanthranoyl)-AMP (Mant-AMP), reveal that nucleotide binding to mtCBS-PPase involves a three-step increase in Mant-AMP fluorescence with relaxation times from 0.01 to 100 s, implying conformational changes in the complex. This effect is reversed by AMP. Metal cofactors (Co(2+) and Mg(2+)) enhance the fluorescence signal but are not absolutely required, unlike what is seen when the catalytic reaction is examined. The relaxation times and amplitudes of the fluorescence signals depend on Mant-AMP concentration in a manner suggestive of the presence of a second binding site for Mant-AMP on the protein. Equilibrium fluorescence titration experiments additionally support the presence of two types of AMP binding sites with different affinities, whereas equilibrium dialysis and membrane filtration measurements reveal binding of one AMP molecule per enzyme monomer, implying negative cooperativity in nucleotide binding. The substrate (PP(i)) modulates Mant-AMP binding, leading to a further conformational change in the enzyme-Mant-AMP complex, and stimulates mtCBS-PPase in alkaline medium within a time scale of minutes, via conversion to a more active form. This active form initially comprises only a third of the enzyme, as estimated from kinetic titration with ADP. AMP inhibits both enzyme forms but is unable to independently induce interconversion. Our results collectively suggest that nucleotides and the substrate induce multiple conformational changes in mtCBS-PPase occurring over a wide time scale; the changes are distinct and almost independent.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Jämsen J,Baykov AA,Lahti R

doi

10.1021/bi9019737

subject

Has Abstract

pub_date

2010-02-09 00:00:00

pages

1005-13

issue

eissn

0006-2960

issn

1520-4995

journal_volume

pub_type

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Nucleotide- and substrate-induced conformational transitions in the CBS domain-containing pyrophosphatase of Moorella thermoacetica.