Structural and kinetic features of family I inorganic pyrophosphatase from Vibrio cholerae.
In this paper, kinetic properties of a soluble inorganic pyrophosphatase of family I from Vibrio cholerae (V-PPase), intestinal pathogen and causative agent of human cholera, are characterized in detail, and the crystal structure of a metal-free enzyme is reported. Hydrolytic activity of V-PPase has been studied as a function of pH, concentration of metal cofactors (Mg2+ or Mn2+), and ionic strength. It has been found that, despite the high conservation of amino acid sequences for the known bacterial PPases of family I, V-PPase differs from the other enzymes of the same family in a number of parameters. Dissociation constants of V-PPase complexed with Mg2+ or Mn2+ were essentially the same as for Escherichia coli PPase (E-PPase). However, the pH optimum of MgPP(i) hydrolysis by V-PPase was shifted to more alkaline pH due to higher values of the pK(a) of ionizable groups for both the free enzyme and the enzyme-substrate complex. The stability of a hexameric form of V-PPase has been studied as a function of pH. The corresponding pK(a) of a group that controls the stability of the hexamer at pH below 6 (pK(a) = 4.4) was significantly lower than in the other hexameric PPases. The crystal structure reported here is analyzed and compared with the structure of E-PPase. The location of amino acid residues that differ in V-PPase and E-PPase is discussed. Since V-PPase has been found to retain its hydrolytic activity in high ionic strength media, the observed structural and kinetic features are analyzed in view of the possible osmoadaptation of this protein.
Rodina
E V
EV
Chemistry Faculty, Lomonosov Moscow State University, Moscow, 119992, Russia. rodina@belozersky.msu.ru
Samygina
V R
VR
Vorobyeva
N N
NN
Sitnik
T S
TS
Kurilova
S A
SA
Nazarova
T I
TI
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D043564:Inorganic Pyrophosphatase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D014734:Vibrio cholerae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D043564:Inorganic Pyrophosphatase