title: Structural and functional consequences of substitutions at the tyrosine 55-lysine 104 hydrogen bond in Escherichia coli inorganic pyrophosphatase.
abstract: Tyrosine 55 and lysine 104 are evolutionarily conserved residues that form a hydrogen bond in the active site of Escherichia coli inorganic pyrophosphatase (E-PPase). Here we used site-directed mutagenesis to examine their roles in structure stabilization and catalysis. Though these residues are not part of the subunit interface, Y55F and K104R (but not K104I) substitutions markedly destabilize the hexameric structure, allowing dissociation into active trimers on dilution. A K104I variant is nearly inactive while Y55F and K104R variants exhibit appreciable activity and require greater concentrations of Mg2+ and higher pH for maximal activity. The effects on activity are explained by (a) increased pK(a)s for the catalytically essential base and acid at the active site, (b) decreases in the rate constant for substrate (dimagnesium pyrophosphate) binding to enzyme-Mg2 complex vs enzyme-Mg3 complex, and (c) parallel decreases in the catalytic constant for the resulting enzyme-Mg2-substrate and enzyme-Mg3-substrate complexes. The results are consistent with the major structural roles of Tyr55 and Lys104 in the active site. The microscopic rate constant for PPi hydrolysis on either the Y55F or K104R variants increases, by a factor of 3-4 in the pH range 7.2-8.0, supporting the hypothesis that this reaction step depends on an essential base within the enzyme active site.
authors.affiliation: A.N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Russia.
authors.forename: I P
authors.initials: IP
authors.lastname: Fabrichniy
authors.forename: V N
authors.initials: VN
authors.lastname: Kasho
authors.forename: T
authors.initials: T
authors.lastname: Hyytiä
authors.forename: T
authors.initials: T
authors.lastname: Salminen
authors.forename: P
authors.initials: P
authors.lastname: Halonen
authors.forename: V Y
authors.initials: VY
authors.lastname: Dudarenkov
authors.forename: P
authors.initials: P
authors.lastname: Heikinheimo
authors.forename: V Y
authors.initials: VY
authors.lastname: Chernyak
authors.forename: A
authors.initials: A
authors.lastname: Goldman
authors.forename: R
authors.initials: R
authors.lastname: Lahti
authors.forename: B S
authors.initials: BS
authors.lastname: Cooperman
authors.forename: A A
authors.initials: AA
authors.lastname: Baykov
mesh_terms: D001704:Biopolymers; D004926:Escherichia coli / Q000201:enzymology*; D006860:Hydrogen Bonding; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D011485:Protein Binding; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D001704:Biopolymers; D014443:Tyrosine; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008274:Magnesium; D008239:Lysine