Metal-free PPi activates hydrolysis of MgPPi by an Escherichia coli inorganic pyrophosphatase.
Soluble inorganic pyrophosphatase from Escherichia coli (E-PPase) is a hexamer forming under acidic conditions the active trimers. We have earlier found that the hydrolysis of a substrate (MgPP(i)) by the trimers as well as a mutant E-PPase Asp26Ala did not obey the Michaelis-Menten equation. To explain this fact, a model has been proposed implying the existence of, aside from an active site, an effector site that can bind PP(i) and thus accelerate MgPP(i) hydrolysis. In this paper, we demonstrate that the noncompetitive activation of MgPP(i) hydrolysis by metal-free PP(i) can also explain kinetic features of hexameric forms of both the native enzyme and the specially obtained mutant E-PPase with a substituted residue Glu145 in a flexible loop 144-149. Aside from PP(i), its non-hydrolyzable analog methylene diphosphonate can also occupy the effector site resulting in the acceleration of the substrate hydrolysis. Our finding that two moles of [32P]PP(i) can bind with each enzyme subunit is direct evidence for the existence of the effector site in the native E-PPase.
Vainonen
Ju P
JP
Chemistry Faculty, Lomonosov Moscow State University, Moscow 119992, Russia.
Vorobyeva
N N
NN
Rodina
E V
EV
Nazarova
T I
TI
Kurilova
S A
SA
Skoblov
Ju S
JS
Avaeva
S M
SM
D000495:Allosteric Site; D011756:Diphosphates / Q000378:metabolism* / Q000494:pharmacology*; D004164:Diphosphonates / Q000494:pharmacology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D017616:Magnesium Compounds / Q000378:metabolism*; D008956:Models, Chemical; D009097:Multienzyme Complexes; D011485:Protein Binding
D011756:Diphosphates; D004164:Diphosphonates; D017616:Magnesium Compounds; D009097:Multienzyme Complexes; C027474:methylene diphosphonate; C034732:magnesium pyrophosphate; D043564:Inorganic Pyrophosphatase