Two-metal Ion Research Articles

The R78K and D117E active-site variants of Saccharomyces cerevisiae soluble inorganic pyrophosphatase: structural studies and mechanistic implications

We have solved the structure of two active-site variants of soluble inorganic pyrophosphatases (PPase), R78K and D117K, at resolutions of 1.85 and 2.15 Å and R-factors of 19.5% and 18.3%, respectively.In the R78K variant structure, the high-affinity phosphate group (P1) is missing, consistent with the wild-type structure showing a bidentate interaction between P1 and Arg78, and solution data showing a decrease in P1 affinity in the variant. The structure explains why the mutation affects P1 and pyrophosphate binding much more than would be expected by the loss of one hydrogen bond: Lys78 forms an ion-pair with Asp71, precluding an interaction with P1. The R78K variant also provides the first direct evidence that the low-affinity phosphate group (P2) can adopt the structure that we believe is the immediate product of hydrolysis, with one of the P2 oxygen atoms co-ordinated to both activating metal ions (M1 and M2). If so, the water molecule (Wat1) between M1 and M2 in wild-type PPase is, indeed, the attacking nucleophile.The D117E variant structure likewise supports our model of catalysis, as the Glu117 variant carboxylate group is positioned where Wat1 is in the wild-type: the potent Wat1 nucleophile is replaced by a carboxylate co-ordinated to two metal ions. Alternative confirmations of Glu117 may allow Wat1 to be present but at much reduced occupancy, explaining why the pKa of the nucleophile increases by three pH units, even though there is relatively little distortion of the active site.These new structures, together with parallel functional studies measuring catalytic efficiency and ligand (metal ion, PPi and Pi) binding, provide strong evidence against a proposed mechanism in which Wat1 is considered unimportant for hydrolysis. They thus support the notion that PPase shares mechanistic similarity with the “two-metal ion” mechanism of polymerases.

Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes

New levels in the understanding of DNA replication have been achieved from recent crystal structure determinations of several DNA polymerases and their substrate complexes. The structure of an α family DNA polymerase from bacteriophage RB69 shows some similarities, but also considerable differences in structure and organization from the pol I family DNA polymerases. Also, the functions of three polymerase domains and their conserved residues have been clarified by studying structures of pol I family DNA polymerases complexed to their substrates. These structures also confirm that an identical two-metal ion catalytic mechanism proposed previously is used by both the nonhomologous pol I and pol β family DNA polymerases.

A two-metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate.

Evidence for a two-metal ion mechanism for cleavage of the HH16 hammerhead ribozyme is provided by monitoring the rate of cleavage of the RNA substrate as a function of La3+ concentration in the presence of a constant concentration of Mg2+. We show that a bell-shaped curve of cleavage activation is obtained as La3+ is added in micromolar concentrations in the presence of 8 mM Mg2+, with a maximal rate of cleavage being attained in the presence of 3 microM La3+. These results show that two-metal ion binding sites on the ribozyme regulate the rate of the cleavage reaction and, on the basis of earlier estimates of the Kd values for Mg2+ of 3.5 mM and > 50 mM, that these sites bind La3+ with estimated Kd values of 0.9 and > 37.5 microM, respectively. Furthermore, given the very different effects of these metal ions at the two binding sites, with displacement of Mg2+ by La3+ at the stronger (relative to Mg2+) binding site activating catalysis and displacement of Mg2+ by La3+ at the weaker (relative to Mg2+) (relative to Mg2+) binding site inhibiting catalysis, we show that the metal ions at these two sites play very different roles. We argue that the metal ion at binding site 1 coordinates the attacking 2'-oxygen species in the reaction and lowers the pKa of the attached proton, thereby increasing the concentration of the attacking alkoxide nucleophile in an equilibrium process. In contrast, the role of the metal ion at binding site 2 is to catalyze the reaction by absorbing the negative charge that accumulates at the leaving 5'-oxygen in the transition state. We suggest structural reasons why the Mg(2+)-La3+ ion combination is particularly suited to demonstrating these different roles of the two-metal ions in the ribozyme cleavage reaction.

The structural basis for pyrophosphatase catalysis.

Soluble inorganic pyrophosphatase (PPase), an essential enzyme central to phosphorus metabolism, catalyzes the hydrolysis of the phosphoanhydride bond in inorganic pyrophosphate. Catalysis requires divalent metal ions which affect the apparent pKas of the essential general acid and base on the enzyme, and the pKa of the substrate. Three to five metal ions are required for maximal activity, depending on pH and enzyme source. A detailed understanding of catalysis would aid both in understanding the nature of biological mechanisms of phosphoryl transfer, and in understanding the role of divalent cations. Without a high-resolution complex structure such a model has previously been unobtainable. We report the first two high-resolution structures of yeast PPase, at 2.2 and 2.0 A resolution with R factors of around 17%. One structure contains the two activating metal ions; the other, the product (MnPi)2 as well. The latter structure shows an extensive network of hydrogen bond and metal ion interactions that account for virtually every lone pair on the product phosphates. It also contains a water molecule/hydroxide ion bridging two metal ions and, uniquely, a phosphate bound to four Mn2+ ions. Our structure-based model of the PPase mechanism posits that the nucleophile is the hydroxide ion mentioned above. This aspect of the mechanism is formally analogous to the "two-metal ion' mechanism of alkaline phosphatase, exonucleases and polymerases. A third metal ion coordinates another water molecule that is probably the required general acid. Extensive Lewis acid coordination and hydrogen bonds provide charge shielding of the electrophile and lower the pKa of the leaving group. This "three-metal ion' mechanism is in detail different from that of other phosphoryl transfer enzymes, presumably reflecting how ancient the reaction is.

One- and Two-Metal Ion Catalysis of the Hydrolysis of Adenosine 3‘-Alkyl Phosphate Esters. Models for One- and Two-Metal Ion Catalysis of RNA Hydrolysis

Adenosine 3‘-O(PO2-)OCH2R phosphate esters have been synthesized with R = 8-hydroxyquinol-2-yl (1a) and 8-(hydroxyquinolyl)-2-methylene (1b). The adenosine 3‘-O(PO2-)OCH2R structure has the essential features of an RNA dinucleotide. Equilibrium binding studies with metal ions Mg2+, Zn2+, Cu2+, and La3+ have been carried out with 1a, 1b, HOCH2R (7a and 7b), and 8-hydroxyquinoline (8), and equilibrium constants (Kas) have been determined for the formation of 1:1 (L)Mn+ complexes. The hydrolysis of 1a and 1b as well as (1a)Mn+ and (1b)Mn+ species are first order in HO-. The rate enhancement for hydrolysis of 1a by complexation with metal ions is as follows: ∼105 with Zn2+, ∼103 with Mg2+, ∼105 with Cu2+, and ∼109 with La3+. Molecular modeling indicates that metal ions ligated to the 8-hydroxyquinoline moiety in the complexes (1a)Mn+ and (1b)Mn+ catalyze 1a and 1b hydrolysis by interacting as Lewis acid catalysts with the negatively charged oxygen atom of the phosphate group. In the instance of La3+ complexe...

Two-metal ion mechanism of bovine lens leucine aminopeptidase: active site solvent structure and binding mode of L-leucinal, a gem-diolate transition state analogue, by X-ray crystallography.

The three-dimensional structures of bovine lens leucine aminopeptidase (blLAP) complexed with L-leucinal and of the unliganded enzyme have been determined at crystallographic resolutions of 1.9 and 1.6 A, respectively. Leucinal binds as a hydrated gem-diol to the active site of b1LAP), resembling the presumed gem-diolated intermediate in the catalytic pathway. One hydroxyl group bridges the two active site metal ions, and the other OH group is coordinated to Zn1. The high-resolution structure of the unliganded enzyme reveals one metal-bound water ligand, which is bridging both zinc ions. Together, these structures support a mechanism in which the bridging water ligand is the attacking hydroxide ion nucleophile. The gem-diolate intermediate is probably stabilized by four coordinating bonds to the dizinc center and by interaction with Lys-262 and Arg-336. In the mechanism, Lys-262 polarizes the peptide carbonyl group, which is also coordinated to Zn1. The Arg-336 side chain interacts with the substrate and the gem-diolate intermediate via water molecules. Near Arg-336 in the b1LAP-leucinal structure, an unusually short hydrogen bond is found between two active site water molecules.

Charged with meaning: the structure and mechanism of phosphoprotein phosphatases

The new structural data obtained for PP1 and calcineurin—A resolve many of the outstanding questions about the catalytic nature of the Ser/Thr phosphatases. These enzymes may now be firmly classed as two-metal ion phosphatases. These data also provide a model for understanding a variety of phosphatases that share the two-metal-ion binding signature motif found in the Ser/Thr phosphatases. These enzymes are clearly important in the control of a variety of cellular functions and in a number of cases are important targets for therapeutic intervention. The structural data obtained for PP1 and calcineurin—A should certainly facilitate efforts to understand, and design inhibitors of, these proteins and related phosphatases.

Two-metal ion mechanism of leucine aminopeptidase. Binding of GEM-diolate transition state analogues

Two-metal ion mechanism of leucine aminopeptidase. Binding of GEM-diolate transition state analogues

Transition state analogue L-leucinephosphonic acid bound to bovine lens leucine aminopeptidase: X-ray structure at 1.65 A resolution in a new crystal form.

The three-dimensional structure of bovine lens leucine aminopeptidase (blLAP) complexed with L-Leucinephosphonic acid (LeuP) has been determined by molecular replacement using the structure of native blLAP as a starting model. Cocrystallization of the enzyme with the inhibitor yielded a new crystal form of space group P321 which has cell dimensions a = 130.4 A and c = 125.4 A. Refinement of the model against data from 7.0 to 1.65 A resolution resulted in a final structure with a crystallographic residual of 0.160 (R(free) = 0.191). The N-terminal amino group of LeuP is coordinated to Zn-489, one phosphoryl oxygen atom bridges both metal ions, and another phosphoryl oxygen atom is coordinated to Zn-488. The side chain of Arg-336 interacts with the inhibitor via three water molecules. LeuP resembles the presumed tetrahedral gem-diolate transition state after direct attack of a water or hydroxide ion nucleophile on the scissile peptide bond. On the basis of the LeuP binding mode and the previous structural and biochemical data, three plausible reaction pathways are evaluated. The two-metal ion mechanisms discussed herein share as common features a metal-bound hydroxide ion nucleophile and polarization of the carbonyl group by the zinc ions. Possible catalytic roles of Arg-336 and Lys-262 in the direct or indirect (through H2O) protonation of the leaving group, in the stabilization of a zinc-bound OH- nucleophile and in the stabilization of the negatively charged intermediate, are discussed. A site 3 metal ion approximately 12 A away from the active site 2 zinc ion probably serves a structural role.

Zinc co-ordination in the DNA-binding domain of the yeast transcriptional activator PPR1

The structure of the native zinc form of the DNA binding domain in the yeast transcriptional activator PPR1 was investigated by extended X-ray absorption fine structure (EXAFS). By carrying out the EXAFS measurements at 11k we were able to demonstrate explicitly the proximity of the two zinc ions ( ZnZn distance = 3.16 ± 0.03 A ̊ ) and the presence of bridging cysteine ligands. The results show that the six cysteine residues co-ordinate two zinc ions in a two-metal ion cluster. PPR1 is the first member of this class of protein for which such information has been obtained.

Structures of Ternary Complexes of Rat DNA Polymerase β, a DNA Template-Primer, and ddCTP

Two ternary complexes of rat DNA polymerase beta (pol beta), a DNA template-primer, and dideoxycytidine triphosphate (ddCTP) have been determined at 2.9 A and 3.6 A resolution, respectively. ddCTP is the triphosphate of dideoxycytidine (ddC), a nucleoside analog that targets the reverse transcriptase of human immunodeficiency virus (HIV) and is at present used to treat AIDS. Although crystals of the two complexes belong to different space groups, the structures are similar, suggesting that the polymerase-DNA-ddCTP interactions are not affected by crystal packing forces. In the pol beta active site, the attacking 3'-OH of the elongating primer, the ddCTP phosphates, and two Mg2+ ions are all clustered around Asp190, Asp192, and Asp256. Two of these residues, Asp190 and Asp256, are present in the amino acid sequences of all polymerases so far studied and are also spatially similar in the four polymerases--the Klenow fragment of Escherichia coli DNA polymerase I, HIV-1 reverse transcriptase, T7 RNA polymerase, and rat DNA pol beta--whose crystal structures are now known. A two-metal ion mechanism is described for the nucleotidyl transfer reaction and may apply to all polymerases. In the ternary complex structures analyzed, pol beta binds to the DNA template-primer in a different manner from that recently proposed for other polymerase-DNA models.

Reaction mechanism of alkaline phosphatase based on crystal structures: Two-metal ion catalysis

Alkaline phosphatase (AP) is a widely distributed non-specific phosphomonoesterase that functions through formation of a covalent phosphoseryl intermediate (E–P). The enzyme also catalyzes phosphoryl transfer reaction to various alcohols. Escherichia coli AP is a homodimer with 449 residues per monomer. It is a metalloenzyme with two Zn 2+ and one Mg 2+ at each active site. The crystal structure of native E. coli AP complexed with inorganic phosphate (P i), which is a strong competitive inhibitor as well as a substrate for the reverse reaction, has been refined at 2·0 Å resolution. Some parts of the molecule have been retraced, starting from the previous 2·8 Å study. The active site has been modified substantially and is described in this paper. The changes in the active site region suggest the need to reinterpret earlier spectral data, and suggestions are made. Also presented are the structures of the Cd-substituted enzyme complexed with inorganic phosphate at 2·5 Å resolution, and the phosphate-free native enzyme at 2·8 Å resolution. At pH 7·5, where the X-ray data were collected, the Cd-substituted enzyme is predominantly the covalent phosphoenzyme (E–P) while the native Zn/Mg enzyme exists in predominantly noncovalent (E · P) form. Implication of these results for the catalytic mechanism of the enzyme is discussed. APs from other sources are believed to function in a similar manner.