Reciprocal effects of substitutions at the subunit interfaces in hexameric pyrophosphatase of Escherichia coli. Dimeric and monomeric forms of the enzyme.
A homohexameric molecule of Escherichia coli pyrophosphatase is arranged as a dimer of trimers, with an active site present in each of its six monomers. Earlier we reported that substitution of His(136) and His(140) in the intertrimeric subunit interface splits the molecule into active trimers (Velichko, I. S., Mikalahti, K., Kasho, V. N., Dudarenkov, V. Y., Hyytiä, T., Goldman, A., Cooperman, B. S., Lahti, R., and Baykov, A. A. (1998) Biochemistry 37, 734-740). Here we demonstrate that additional substitutions of Tyr(77) and Gln(80) in the intratrimeric interface give rise to moderately active dimers or virtually inactive monomers, depending on pH, temperature, and Mg(2+) concentration. Successive dissociation of the hexamer into trimers, dimers, and monomers progressively decreases the catalytic efficiency (by 10(6)-fold in total), and conversion of a trimer into dimer decreases the affinity of one of the essential Mg(2+)-binding sites/monomer. Disruptive substitutions predominantly in the intratrimeric interface stabilize the intertrimeric interface and vice versa, suggesting that the optimal intratrimeric interaction is not compatible with the optimal intertrimeric interaction. Because of the resulting "conformational strain," hexameric wild-type structure appears to be preformed to bind substrate. A hexameric triple variant substituted at Tyr(77), Gln(80), and His(136) exhibits positive cooperativity in catalysis, consistent with this model.
Salminen
A
A
Department of Biochemistry, University of Turku, FIN-20500 Turku, Finland.
Efimova
I S
IS
Parfenyev
A N
AN
Magretova
N N
NN
Mikalahti
K
K
Goldman
A
A
Baykov
A A
AA
Lahti
R
R
D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000235:genetics; D002384:Catalysis; D018360:Crystallography, X-Ray; D019281:Dimerization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D018698:Glutamic Acid / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D017616:Magnesium Compounds / Q000494:pharmacology; D020836:Protein Structure, Quaternary / Q000187:drug effects; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D014443:Tyrosine / Q000235:genetics
D017616:Magnesium Compounds; D005973:Glutamine; D001224:Aspartic Acid; D018698:Glutamic Acid; D014443:Tyrosine; D011755:Pyrophosphatases