Probing Essential Water in Yeast Pyrophosphatase by Directed Mutagenesis and Fluoride Inhibition Measurements

Pohjanjoki, Pekka; Fabrichniy, Igor P.; Kasho, Vladimir N.; Cooperman, Barry S.; Goldman, Adrian; Baykov, Alexander A.; Lahti, Reijo

doi:10.1074/jbc.m007360200

Cited by 26 publications

(40 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The flexibility of the protein structure allows for efficient metal binding in the variant enzymes, but the bulk of the resulting complex is non-productive because the metal is mispositioned. Notably, the lack of effect of similar substitutions (Asp 3 Glu) of metal ligands on thermodynamically controlled metal binding is well documented in soluble PPases (34,35).…”

Section: Discussionmentioning

confidence: 99%

Elucidating the Role of Conserved Glutamates in H+-pyrophosphatase of Rhodospirillum rubrum

Malinen

Belogurov

Salminen

et al. 2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

H ؉ -pyrophosphatase (H ؉ -PPase) catalyzes pyrophosphate-driven proton transport against the electrochemical potential gradient in various biological membranes. All 50 of the known H ؉ -PPase amino acid sequences contain four invariant glutamate residues. In this study, we use site-directed mutagenesis in conjunction with functional studies to determine the roles of the glutamate residues Glu 197 , Glu 202 , Glu 550 , and Glu 649 in the H ؉ -PPase of Rhodospirillum rubrum (R-PPase). All residues were replaced with Asp and Ala. The resulting eight variant R-PPases were expressed in Escherichia coli and isolated as inner membrane vesicles. All substitutions, except E202A, generated enzymes capable of PP i hydrolysis and PP i -energized proton translocation, indicating that the negative charge of Glu 202 is essential for R-PPase function. The hydrolytic activities of all other PPase variants were impaired at low Mg 2؉ concentrations but were only slightly affected at high Mg 2؉ concentrations, signifying that catalysis proceeds through a three-metal pathway in contrast to wild-type R-PPase, which employs both two-and three-metal pathways. Substitution of Glu 197 , Glu 202 , and Glu 649 resulted in decreased binding affinity for the substrate analogues aminomethylenediphosphonate and methylenediphosphonate, indicating that these residues are involved in substrate binding as ligands for bridging metal ions. Following the substitutions of Glu 550 and Glu 649 , R-PPase was more susceptible to inactivation by the sulfhydryl reagent mersalyl, highlighting a role of these residues in maintaining enzyme tertiary structure. None of the substitutions affected the coupling of PP i hydrolysis to proton transport.

show abstract

Section: Discussionmentioning

confidence: 99%

Elucidating the Role of Conserved Glutamates in H+-pyrophosphatase of Rhodospirillum rubrum

Malinen

Belogurov

Salminen

et al. 2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…This would be expected if F Ϫ replaced H 2 O, with concomitant loss of hydrogen bond donors and increase in negative charge. Second, fluoride inhibition data (12) suggest that F Ϫ binds at this position, and, third, in the D117E variant (5) a carboxylate oxygen of E117 also binds here.…”

Section: Refinement and Selection Of Structures For Comparisonmentioning

confidence: 99%

Toward a quantum-mechanical description of metal-assisted phosphoryl transfer in pyrophosphatase

Heikinheimo

Tuominen

Ahonen

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

102

125

View full text Add to dashboard Cite

The wealth of kinetic and structural information makes inorganic pyrophosphatases (PPases) a good model system to study the details of enzymatic phosphoryl transfer. The enzyme accelerates metal-complexed phosphoryl transfer 10 10 -fold: but how? Our structures of the yeast PPase product complex at 1.15 Å and fluoride-inhibited complex at 1.9 Å visualize the active site in three different states: substrate-bound, immediate product bound, and relaxed product bound. These span the steps around chemical catalysis and provide strong evidence that a water molecule (O nu) directly attacks PPi with a pK a vastly lowered by coordination to two metal ions and D117. They also suggest that a low-barrier hydrogen bond (LBHB) forms between D117 and O nu, in part because of steric crowding by W100 and N116. Direct visualization of the double bonds on the phosphates appears possible. The flexible side chains at the top of the active site absorb the motion involved in the reaction, which may help accelerate catalysis. Relaxation of the product allows a new nucleophile to be generated and creates symmetry in the elementary catalytic steps on the enzyme. We are thus moving closer to understanding phosphoryl transfer in PPases at the quantum mechanical level. Ultra-high resolution structures can thus tease out overlapping complexes and so are as relevant to discussion of enzyme mechanism as structures produced by time-resolved crystallography. Inorganic pyrophosphatases (PPases) catalyze one of the oldest and most common reactions in cells and provide a good system for detailed analysis of enzymatic phosphoryl transfer from polyphosphate to water. The kinetics are well characterized (1, 2) and high-resolution structures are available along the reaction pathway (3). The enzyme accelerates hydrolysis of metal complexed inorganic pyrophosphate by 10 10 compared with the uncatalyzed reaction (1)-but for PPases, as for enzymes in general, the exact source of rate enhancement remains unclear.The original model of catalysis suggested that the mechanism proceeded in four steps with all steps after substrate binding partially rate-determining (1). The nucleophile, which is generated by coordinating a water molecule (O nu ) to two metal ions and which is further strengthened by donating a hydrogen bond to D117, is one key to pyrophosphate hydrolysis in PPases. In addition, the substrate pK a is adjusted by extensive coordination to charged atoms (positively charged side chains and M 2ϩ ; ref.3).Our most recent solution studies (P. Halonen, unpublished data; refs. 2 and 4) indicate that the enzyme-substrate complex (EM 2 :MPPi or EM 2 :M 2 PPi) undergoes isomerization during the catalytic cycle (Scheme 1; ref. 4). In addition, fluoride inhibition studies (4) are consistent with structural studies (3,5) suggesting that the nucleophile is coordinated to D117.We earlier determined the structure of complexes A and E (Scheme 1), but now have direct structural information on the mechanistically key intermediates C and D, as well as much higher res...

show abstract

“…Accordingly, all our experiments were performed at pH 7.5. The yeast PPase is inhibited by NaF (26,27), and in the conditions we used, half-maximal inhibition was attained in the presence of 0.12 mM NaF (Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

“…During catalysis the enzyme-substrate complex undergoes isomerization; one of the two water molecules bound to Mg 2ϩ dissociates from the enzyme-substrate complex, and the second water molecule is used to hydrolyze PP i (26, 27, 46 -48). Inhibition by NaF is promoted by replacement of the magnesium-bound water molecule by F Ϫ (26,27,47). According to the early measurements (40 -43), conversion of PP i from high into low energy should occur during isomerization of the enzyme.…”

Section: Energy Of Hydrolysis Of Phosphate Compounds During Catalysismentioning

confidence: 99%

Heat of PPi Hydrolysis Varies Depending on the Enzyme Used

da‐Silva¹,

Bomfim²,

Galina³

et al. 2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

With yeast-soluble inorganic pyrophosphatase, the heat released during PP i hydrolysis was ؊6.3 kcal/mol regardless of the KCl concentration in the medium. With the membrane-bound pyrophosphatase of corn vacuoles, the heat released varies between ؊23.5 and ؊7.5 kcal/mol depending on the KCl concentration in the medium and whether or not a H ؉ gradient is formed across the vacuole membranes. The data support the proposal that enzymes are able to handle the energy derived from phosphate compound hydrolysis in such a way as to determine the parcel that is used for work and the fraction that is converted into heat (de Meis, L. (2001) J. Biol. Chem. 276, 25078 -25087).Evidence reported in the past 5 years indicates that enzymes are able to handle the energy derived from the hydrolysis of phosphate compounds in such a way as to determine the parcel that is used for work and the fraction that is converted into heat (1-12). The ability to modulate the conversion of energy into either heat or work varies depending on both the enzyme and the experimental conditions used. This was first observed with the sarco/endoplasmic reticulum Ca 2ϩ -ATPases (SERCA), 1 a family of membrane-bound ATPases that are able to translocate Ca 2ϩ ion across the membrane by using the chemical energy derived from ATP hydrolysis. With these enzymes it was found that the heat released during ATP hydrolysis may vary from 10 to 30 kcal/mol depending on the SERCA isoform used and on whether or not a Ca 2ϩ gradient is formed across the membrane (1-10). Kinetic measurements indicate that the SERCA are able to hydrolyze ATP through two catalytic routes (4 -6, 8, 13-15). In one of them hydrolysis is coupled with the translocation of Ca 2ϩ through the membrane. In this case, a part of the chemical energy derived from ATP cleavage is used for Ca 2ϩ transport, and a part is converted into heat. The second route is a shortcut of the transport cycle where the cleavage of ATP is completed in a step that precedes the translocation of Ca 2ϩ through the membrane, and all the energy derived from ATP hydrolysis is converted into heat (4,5,8,10,(13)(14)(15). Thus, during the course of the reaction, the amount of heat released varies depending on how much ATP is cleaved in each of these two routes.Recently, Bianconi (11) found that the enthalpy for the reactions of yeast hexokinase varies depending on the isozymes used, and Szöke et al. (12) in an elegant theoretical analysis proposed that enzyme could adjust the reaction path by altering the reversible interchange of different forms of energies (chemical, electrical, and mechanical). In these views, the same reaction catalyzed by different enzymes would lead to the release of different amounts of heat. In the report by Szöke et al. (12), it was proposed that the amount of heat produced during PP i hydrolysis by the soluble and membrane-bound PPases should be markedly different. The soluble PPase would convert ⌬G into heat, whereas for the membrane-bound PPases, less heat should be released because part of the ener...

show abstract

Probing Essential Water in Yeast Pyrophosphatase by Directed Mutagenesis and Fluoride Inhibition Measurements

Cited by 26 publications

References 37 publications

Elucidating the Role of Conserved Glutamates in H+-pyrophosphatase of Rhodospirillum rubrum

Elucidating the Role of Conserved Glutamates in H+-pyrophosphatase of Rhodospirillum rubrum

Toward a quantum-mechanical description of metal-assisted phosphoryl transfer in pyrophosphatase

Heat of PPi Hydrolysis Varies Depending on the Enzyme Used

Contact Info

Product

Resources

About