Trimeric inorganic pyrophosphatase of Escherichia coli obtained by directed mutagenesis.
Escherichia coli inorganic pyrophosphatase is a tight hexamer of identical subunits. Replacement of both His136 and His140 by Gln in the subunit interface results in an enzyme which is trimeric up to 26 mg/mL enzyme concentration in the presence of Mg2+, allowing direct measurements of Mg2+ binding to trimer by equilibrium dialysis. The results of such measurements, together with the results of activity measurements as a function of [Mg2+] and pH, indicate that Mg2+ binds more weakly to one of the three sites per monomer than it does to the equivalent site in the hexamer, suggesting this site to be located in the trimer:trimer interface. The otherwise unobtainable hexameric variant enzyme readily forms in the presence of magnesium phosphate, the product of the pyrophosphatase reaction, but rapidly dissociates on dilution into medium lacking magnesium phosphate or pyrophosphate. The kcat values are similar for the variant trimer and hexamer, but Km values are 3 orders of magnitude lower for the hexamer. Thus, while stabilizing hexamer, the two His residues, per se, are not absolutely required for active-site structure rearrangement upon hexamer formation. The reciprocal effect of hexamerization and product binding to the active site is explained by destabilization of alpha-helix A, contributing both to the active site and the subunit interface.
Velichko
I S
IS
A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow 119899, Russia.
Mikalahti
K
K
Kasho
V N
VN
Dudarenkov
V Y
VY
Hyytiä
T
T
Goldman
A
A
Cooperman
B S
BS
Lahti
R
R
Baykov
A A
AA
D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D005973:Glutamine / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D017616:Magnesium Compounds / Q000494:pharmacology; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017616:Magnesium Compounds; D010710:Phosphates; D005973:Glutamine; C030781:magnesium phosphate; D006639:Histidine; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase