Mechanism of Ca2+-induced inhibition of Escherichia coli inorganic pyrophosphatase.
The causes of inhibition of Escherichia coli inorganic pyrophosphatase (PPase) by Ca2+ were investigated. The interactions of several mutant pyrophosphatases with Ca2+ in the absence of substrate were analyzed by equilibrium dialysis. The kinetics of Ca2+ inhibition of hydrolysis of the substrates MgPPi and LaPPi by the native PPase and three mutant enzymes (Asp-42-Asn, Ala, and Glu) were studied. X-Ray data on E. coli PPase complexed with Ca2+ or CaPPi solved at atomic resolution were analyzed. It was shown that, in the course of the catalytic reaction, Ca2+ replaces Mg2+ at the M2 site, which shows higher affinity for Ca2+ than for Mg2+. Different properties of these cations account for active site deformation. Our findings indicate that the filling of the M2 site with Ca2+ is sufficient for PPase inhibition. This fact proves that Ca2+ is incapable of properly activating the H2O molecule for nucleophilic attack on PPi. It was also demonstrated that Ca2+, as a constituent of the non-hydrolyzable substrate analog CaPPi, competes with MgPPi at the M3 binding site. As a result, Ca2+ is a powerful inhibitor of all known PPases. Other possible reasons for the inhibitory effect of Ca2+ on the enzyme activity are also considered.
Avaeva
S M
SM
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119899, Russia. avaeva@libro.genebee.msu.su
Vorobyeva
N N
NN
Kurilova
S A
SA
Nazarova
T I
TI
Polyakov
K M
KM
Rodina
E V
EV
Samygina
V R
VR
D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution; D001216:Asparagine / Q000378:metabolism; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D003956:Dialysis; D011756:Diphosphates / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D018698:Glutamic Acid / Q000378:metabolism; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D007811:Lanthanum / Q000737:chemistry; D017616:Magnesium Compounds / Q000378:metabolism; D008958:Models, Molecular; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
D011756:Diphosphates; D017616:Magnesium Compounds; D001224:Aspartic Acid; D018698:Glutamic Acid; D007811:Lanthanum; D001216:Asparagine; C034732:magnesium pyrophosphate; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D000409:Alanine; D002118:Calcium