Metal cofactors play a dual role in Mycobacterium tuberculosis inorganic pyrophosphatase.
Inorganic pyrophosphatase from Mycobacterium tuberculosis (Mt-PPase) is one of the possible targets for the rational design of anti-tuberculosis agents. In this paper, functional properties of this enzyme are characterized in the presence of the most effective activators--Mg2+ and Mn2+. Dissociation constants of Mt-PPase complexed with Mg2+ or Mn2+ are essentially similar to those of Escherichia coli PPase. Stability of a hexameric form of Mt-PPase has been characterized as a function of pH both for the metal-free enzyme and for Mg2+- or Mn2+-enzyme. Hexameric metal-free Mt-PPase has been shown to dissociate, forming monomers at pH below 4 or trimers at pH from 8 to 10. Mg2+ or Mn2+ shift the hexamer-trimer equilibrium found for the apo-Mt-PPase at pH 8-10 toward the hexameric form by stabilizing intertrimeric contacts. The pK(a) values have been determined for groups that control the observed hexamer-monomer (pK(a) 5.4), hexamer-trimer (pK(a) 7.5), and trimer-monomer (pK(a) 9.8) transitions. Our results demonstrate that due to the non-conservative amino acid residues His21 and His86 in the active site of Mt-PPase, substrate specificity of this enzyme, in contrast to other typical PPases, does not depend on the nature of the metal cofactor.
Rodina
E V
EV
Chemical Faculty, Lomonosov Moscow State University, Moscow, Russia. rodina@belozersky.msu.ru
Vainonen
L P
LP
Vorobyeva
N N
NN
Kurilova
S A
SA
Sitnik
T S
TS
Nazarova
T I
TI
D000995:Antitubercular Agents / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism*; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D043564:Inorganic Pyrophosphatase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008670:Metals / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D003067:Coenzymes; D008670:Metals; D021122:Protein Subunits; D043564:Inorganic Pyrophosphatase