Harold W. Wyckoff

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 Zn2+ and one Mg2+ at each active site. The crystal structure of native E. coli AP complexed with inorganic phosphate (Pi), which is a strong competitive inhibitor as well as a substrate for the reverse reaction, has been refined at 2.0 A resolution. Some parts of the molecular have been retraced, starting from the previous 2.8 A 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 A resolution, and the phosphate-free native enzyme at 2.8 A 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.

24 Bovine Pancreatic Ribonuclease

Publisher Summary This chapter discusses the process of isolation, chromatography, structure, and molecular and catalytic properties of bovine pancreatic ribonuclease. At present there are three simple and widely used chromatographic procedures: (1) Hirs base their method on the carboxyl ion exchange resin IRC-50 with 0.2 M phosphate buffer pH 6.45 as the eluting medium. The principal active component of the enzyme preparation is well retarded and is universally referred to as ribonuclease-A. Several poorly resolved faster running peaks are usually seen, the area having the highest activity and running closest to A normally being called ribonuclease B. The ratio of A to B varies with the preparation but may be as high as 10 to 1. (2) Taborsky has described a system based on carboxymethyl cellulose as the exchanger operated in Tris buffer at pH 8 with a sodium chloride gradient. The excellent and adjustable resolution of this system is frequently useful. The principal peak, labeled D by Taborsky, is indistinguishable from ribonuclease-A in the IRC-50 system. (3) Crestfield found chromatography on sulfoethyl Sephadex valuable. Ribonuclease-A may develop heterogeneity during lyophilization and storage. Aggregation appears to occur. A careful study of the preparation problem has been made by Crestfield by using chromatography on Sephadex G-75, and sulfoethyl Sephadex C-25 as well as IRC-50. These authors recommended that RNase-A be stored as a solution in phosphate buffer at –20°C, that salts be exchanged by dialysis or pre-equilibrated Sephadex columns, and that concentration, if necessary, be effected by ultrafiltration. If lyophilization is necessary, it should be carried out from dilute salt-free solution to minimize aggregate formation. The aggregates can be converted to monomers by heating to 60° for a few minutes at neutral pH. The properties of the ribonuclease dimer are also discussed in the chapter.

Refined structure of alkaline phosphatase from Escherichia coli at 2.8 Å resolution

The structure of alkaline phosphatase from Escherichia coli has been determined to 2.8 A resolution. The multiple isomorphous replacement electron density map of the dimer at 3.4 A was substantially improved by molecular symmetry averaging and solvent flattening. From these maps, polypeptide chains of the dimer were built using the published amino acid sequence. Stereochemically restrained least-squares refinement of this model against native data, starting with 3.4 A data and extending in steps to 2.8 A resolution, proceeded to a final overall crystallographic R factor of 0.256. Alkaline phosphatase-phosphomonoester hydrolase (EC 3.1.3.1) is a metalloenzyme that forms an isologous dimer with two reactive centers 32 A apart. The topology of the polypeptide fold of the subunit is of the alpha/beta class of proteins. Despite the similarities in the overall alpha/beta fold with other proteins, alkaline phosphatase does not have a characteristic binding cleft formed at the carboxyl end of the parallel sheet, but rather an active pocket that contains a cluster of three functional metal sites located off the plane of the central ten-stranded sheet. This active pocket is located near the carboxyl ends of four strands and the amino end of the antiparallel strand, between the plane of the sheet and two helices on the same side. Alkaline phosphatase is a non-specific phosphomonoesterase that hydrolyzes small phosphomonoesters as well as the phosphate termini of DNA. The accessibility calculations based on the refined co-ordinates of the enzyme show that the active pocket barely accommodates inorganic phosphate. Thus, the alcoholic or phenolic portion of the substrate would have to be exposed on the surface of the enzyme. Two metal sites, M1 and M2, 3.9 A apart, are occupied by zinc. The third site, M3, 5 A from site M2 and 7 A from site M1, is occupied by magnesium or, in the absence of magnesium, by zinc. As with other zinc-containing enzymes, histidine residues are ligands to zinc site M1 (three) and to zinc site M2 (one). Ligand assignment and metal preference indicate that the crystallographically found metal sites M1, M2 and M3 correspond to the spectroscopically deduced metal sites A, B and C, respectively. Arsenate, a product analog and enzyme inhibitor, binds between Ser102 and zinc sites M1 and M2. The position of the guanidinium group of Arg 166 is within hydrogen-bonding distance from the arsenate site.(ABSTRACT TRUNCATED AT 400 WORDS)

Structure of alkaline phosphatases.

The crystal structure of alkaline phosphatase (AP) from Escherichia coli, which is a prototype for mammalian APs, has been refined to a crystallographic R-factor of 0.184 at 2.0 A resolution. During the course of the refinement residues 380 to 410 were retraced and 190 to 200 were shifted by one residue, and substantial changes in the active site of the enzyme were made. Based on the refined structure and the sequences of mammalian enzymes (25-30% strict homology) we have modelled the core of the three dimensional structures of the mammalian alkaline phosphatases. Considerable circumstantial evidence suggests that this is valid despite the fact that the mammalian enzymes are larger, contain carbohydrate and are membrane associated through a phosphatidylinositol moiety. The active site of the molecule is highly conserved but specific changes in the secondary ligands to bound phosphate and the Mg metal are observed.

Design of a diffractometer and flow cell system for X-ray analysis of crystalline proteins with applications to the crystal chemistry of ribonuclease-S

A flow cell in which a protein crystal is embedded in a changeable liquid medium during X-ray diffraction studies has proved useful in investigating the crystal chemistry of ribonuclease-S. The cell consists of a small polyethylene tube held in a brass yoke which in turn is held by a standard goniometer head. The crystal is prevented from moving by nesting it in cotton linters or particles of Sephadex. Serious scatter of X-rays from the cell and the liquid is reduced to nominal levels by careful arrangement of the collimating system of the diffractometer and by a special counting procedure which gives tolerance of settings without undue increase of noise level. Applications presented include: studies of diffusion rates of solvent and inhibitors into and out of the crystal with time constants ranging from ninety seconds for ammonium sulfate to many hours for iodinated nucleotides; pH effects on unit-cell constants; curves of binding of iodinated inhibitors as a function of external concentration; comparison of binding sites of various inhibitors; interaction of platinum compounds with the enzyme; catalyzed photo-oxidation of specific residues in situ; and measurements of intensity changes due to heavy-atom binding for use in phasing the protein reflections. The flow cell method is particularly useful in studies where changes of intensity rather than absolute values are of prime importance.

The structure of cytidilyl(2′,5′)adenosine when bound to pancreatic ribonuclease S

Abstract The three-dimensional structure of the RNase S complex with the synthetic dinucleoside monophosphate cytidilyl(2′,5′)adenosine(C2,p5,A) is determined using difference Fourier techniques at 2.0 A resolution in conjunction with computer graphic model-building and energy minimization. The latter has been carried out as a function of the rigid body parameters of the dinucleoside monophosphate and the dihedral angles of the nucleoside portion as well as of relevent amino acids in the active site of the enzyme. The bound dinucleoside monophosphate is found to assume an extended conformation, with the adenine and cytidine bases nearly perpendicular. The bases form specific hydrogen bonds with groups in the active site. Although the atoms involved in the recognition of the pyrimidine base by the enzyme are the same as in the pyrimidine bases of UMP, CMP and UpcA, the details of the binding are different. The adenosine moiety blocks most of the various positions that His119 occupies in the native enzyme and forces it into one well-defined position. One of the His119 ring protons is in contact with O(5′) (the leaving group), O(1′) of the adenine ribose and with a free phosphoryl oxygen. No strong charge contacts with the phosphate group are observed. We show how combining X-ray data with computer graphic model-building, electron density fitting and energy calculations leads to the model we propose and discuss in detail the enzyme-nucleic acid interactions.

Crystal structure of muconolactone isomerase at 3.3 A resolution.

The crystal structure of muconolactone isomerase from Pseudomonas putida, a unique molecule with ten 96 amino acid subunits and 5-fold, and 2-fold symmetries, has been solved at 3.3 A resolution. The non-crystallographic symmetries were used to refine the initial single isomorphous replacement phases and produce an interpretable 10-fold averaged map. The backbone trace is complete and confirmed by the amino acid sequence fit. Each subunit is composed of a body with two alpha-helices and an antiparallel twisted beta-sheet of four strands, and an extended arm. The helices and the sheet fold to form a two-layered structure with an enclosed hydrophobic core and a partially formed putative active site pocket. The C-terminal arm of another subunit related by a local dyad symmetry extends over the core to complete this pocket. The decameric protein is almost spherical, with the helices forming the external coat. There is a large hydrophilic cavity in the center with open ends along the 5-fold axis. Molecular interactions between subunits are extensive. Each subunit contacts four neighbors and loses nearly 40% of its solvent contact area on oligomerization.

Structure of alkaline phosphatase with zinc/magnesium cobalt or cadmium in the functional metal sites

Abstract Crystals of alkaline phosphatase (EC 3.1.3.1; Mr 94,000) grown at pH 9.5 from 2.25 m-(NH4)2SO4 with 5 × 10−5 m-Zn and 10−2 m-Mg present were analyzed by X-ray diffraction at pH 7.5 in 2.66 m-(NH4)2SO4 with 10−2 m-Zn and 10−2 m-Mg present. The crystals are orthorhombic with a = 195.5 A , b = 168.3 A and c = 76.33 A , and the space group is I222. X-ray phases were determined by the multiple isomorphous replacement and anomalous dispersion method using K2PtCl4, KAu(CN)2 and K2OsO4 derivatives. The electron density maps and analysis of metal binding sites reveal one molecule per asymmetric unit with an internal, non-crystallographic, 2-fold rotation axis relating the subunits. Each subunit contains a major α β domain with a seven-stranded β-sheet flanked by helices. The sheets are roughly coplanar but the general direction of the strands in each is at 20 ° to the rotation axis and thus 40 ° from each other. The helical content of the α β domain is approximately 27% of the 459 residues in the monomer and the β content is approximately 7%. The chains in a smaller domain are more convoluted and less easily characterized than in the α β domain. In both there is extensive monomer-monomer contact. Removal of the zinc and magnesium from the parent crystal produces a stable apoenzyme crystal and addition of cobalt at 10−2 m or cadmium at 10−2 or 5 × 10−2 m reveals seven metal binding sites per dimer. The active centers are 32 A apart and each is shown by anomalous dispersion data to contain two metal binding sites, A and B. The cadmium derivative refinement determined the A-B separation to be 4.9 A. Comparison of the parent and apo structures by means of difference maps reveals the double metal site with Zn at A and probably Mg at B. A prominent, partially resolved peak centered 7 A away is interpreted as a stabilization of the backbone in this position by the metal ion co-ordination to a side-chain. Several negative peaks within 10 A of the metals indicate local differences between apo and native structures but no significant differences are seen in the other parts of the molecule. At 5 × 10−2 m-Cd two metal sites (D and D′) are found 25.5 A from the active center, on the surface of the minor domain. They are related to each other by the molecular 2-fold axis with a D-D′ distance of 25 A. The seventh Cd site, E, is 20 A from the active center, on the major domain, near a crystalline contact region, and devoid of any molecular symmetry mate. The apparent dissociation constants for cadmium at the A, B and D sites (and A′, B′, D′) are 3 × 10−3 m, 1.5 × 10−1 m and 1.3 × 10−2 m, respectively. Thus in these conditions cadmium is seen to distribute between A and B sites when the combined stoichiometry is two metal ions per dimer.

A crystallographic study of alkaline phosphatase at 7.7 Å resolution

Abstract An electron density map of crystalline alkaline phosphatase has been calculated to 7.7 A resolution. The enzyme is present as a dimer on the 2-fold symmetry axis at 0, x, 1 6 in space group P3121. An orthorhombic form briefly examined is able to contain a tetrameric molecule with at least 2-fold symmetry. The location and environment of two zinc(II) ions has been determined in the dimer by an EDTA treatment of the crystalline enzyme. A small region of electron density located 10 A from the zinc shifts by 8 A when the metal is removed. The shift is reversed when zinc is added to the apoenzyme. Hg(II) binds preferentially at the vacant zinc site, and Hg(II) will also exchange with the zinc on the untreated enzyme. The slight 2 A displacement of Hg from the zinc position may be related to the known inactivity of the mercury enzyme. The two Zn(II) ions are equivalent by symmetry. They are 32 A apart and 16 A from the dimer axis. This places them within the molecular envelope, but large channels leading inward from opposite sides of the dimer would allow a substrate molecule to come within 3 A of a zinc ion. Also, there is space available for a substrate to lie on the dimer axis between the two ions, though 10 A of protein density would be between the substrate and each zinc ion. In an inconclusive search for active site regions, the enzyme was exposed to p-iodophenylthiophosphate, 2-hydroxy-3-acetomercuri-5-nitrobenzyl-phosphonate, and arsenate ion.

Crystallographic observations of the metal ion triple in the active site region of alkaline phosphatase.

Diffraction analysis reveals three metal ion binding sites, M1, M2 and M3, in each of two symmetric active centers 32 A apart in alkaline phosphatase from Escherichia coli with intermediate distances within the center of 4, 5 and 7 A for M1-M2, M2-M3 and M1-M3, respectively. A fourth site, M4, has been reported 25 A away. Arsenate, a product analog, binds adjacent to M1 and M2. The active serine residue, 102, which is phosphorylated during normal enzymatic turnover, is also adjacent to M1 and M2 and arginine 166 is adjacent to the arsenate. The implication with respect to the mechanism is that M1, M2 and Arg 166 neutralize and redistribute charges within the phosphate group, activate the serine hydroxyl, and stabilize transition states during bond formation and breakage. Three sites, A, B and C, have been deduced from solution studies and defined specifically on the basis of nuclear magnetic resonance data, binding studies and activity data. The evidence suggests correspondence of A to M1, B to M2, and C to M3. Strong antagonism between binding at M1 and M2 is evidenced crystallographically by a pseudo-saturation, which is relieved by phosphate binding. Local destabilization of the protein, particularly residues 323 through 333, is produced by removal of metals from the crystal.