The role of Asp42 in Escherichia coli inorganic pyrophosphatase functioning.
Excess of Mg2+ ions is known to inhibit the soluble inorganic pyrophosphatases (PPases). In contrast, the mutant Escherichia coli inorganic pyrophosphatase Asp42-->Asn is three times more active than native and retains its activity at high Mg2+ concentration. In this paper, another two mutant variants with Asp42 replaced by Ala or Glu were investigated to characterize the role of Asp42 in catalysis. pH-independent kinetic parameters of MgPPi hydrolysis and the dissociation constants for the activating and inhibitory Mg2+ ions were calculated. It was shown that Mg2+ inhibition of MgPPi hydrolysis by native PPase exhibited uncompetitive kinetics under the saturating substrate concentration. All three substitutions of Asp42 lead to a sharp decrease of inhibitory Mg2+ affinity to the enzyme. These findings allow determination of the sites of inhibitory and substrate Mg2+ ions binding to PPase. Common features of these mutants allow the conclusion that the function of Asp42 is to accurately coordinate the residues implicated in the substrate and the inhibitory Mg2+ ion binding to PPase active site. Structural analysis of PPase complexed with Mg2+ compared with PPase complexed with Mn2+ and reaction products confirms this supposition.
Rodina
E V
EV
A.N. Belozersky Institute of Physico-Chemical Biology, Department of Protein Chemistry, Moscow, Russia. rodina@belozersky.msu.ru
Vainonen
Y P
YP
Vorobyeva
N N
NN
Kurilova
S A
SA
Nazarova
T I
TI
Avaeva
S M
SM
D019943:Amino Acid Substitution; D001224:Aspartic Acid; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry
D011994:Recombinant Proteins; D001224:Aspartic Acid; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008274:Magnesium