A site-directed mutagenesis study on Escherichia coli inorganic pyrophosphatase. Glutamic acid-98 and lysine-104 are important for structural integrity, whereas aspartic acids-97 and -102 are essential for catalytic activity.
Analysis of the conservation of functional residues between yeast and Escherichia coli inorganic pyrophosphatases (PPases) suggested that Asp-97, Glu-98, Asp-102, and Lys-104 are important for the action of E. coli PPase [Lahti, R., Kolakowski, L. F., Heinonen, J., Vihinen, M., Pohjanoksa, K., & Cooperman, B. S. (1990) Biochim. Biophys. Acta 1038, 338-345]. We replaced these four residues by oligonucleotide-directed mutagenesis, giving variant PPases DV97, DE97, EV98, DV102, DE102, KI104, and KR104. PPase variants DV97, DV102, and KI104 had no enzyme activity, whereas PPase variants DE97, EV98, DE102, and KR104 had 22%, 33%, 3%, and 3% of the wild-type PPase activity, respectively. This suggests that Asp-97, Asp-102, and Lys-104 are essential for the catalytic activity of E. coli PPase. PPase variants DV98 and KR104 also had an increased sensitivity to heat denaturation; incubation of these mutant PPases at 75 degrees C for 15 min in the presence of 5 mM magnesium ion decreased the activity to 20% and 1%, respectively, of the initial value while 74% of the activity was observed with wild-type PPase. Furthermore, these thermolabile mutant PPases displayed the most profound conformational changes of the PPase variants examined, as demonstrated by the binding of the fluorescent dye Nile red that monitors the hydrophobicity of protein surfaces. Accordingly, Glu-98 and Lys-104 seem to be important for the structural integrity of E. coli PPase.
Lahti
R
R
Department of Biochemistry, University of Turku, Finland.
Pohjanoksa
K
K
Pitkäranta
T
T
Heikinheimo
P
P
Salminen
T
T
Meyer
P
P
Heinonen
J
J
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005971:Glutamates / Q000235:genetics; D018698:Glutamic Acid; D006358:Hot Temperature; D043564:Inorganic Pyrophosphatase; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D011755:Pyrophosphatases / Q000235:genetics*; D012995:Solubility; D013329:Structure-Activity Relationship
D004269:DNA, Bacterial; D005971:Glutamates; D001224:Aspartic Acid; D018698:Glutamic Acid; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008239:Lysine