The volume and structure of the chymotrypsin active site

It was found that the chymotrypsin active site is located in the largest cleft on the enzyme surface approximated by a sphere with a radius of 20 angstroms. The active site cleft volume is about 2 nm3, as computed by the Monte-Carlo method. The size and shape of the active site cleft-- the intersection of two unequal spheres--are sufficient for large (about 1 nm3) fragments of substrate molecules to enter the active site. The active site bottom and the adjacent narrow section are about 600 angstroms3 in volume and may serve as a combustion chamber of a water-substrate mixture during the operation of the enzyme machinery. Intrinsic water molecules inside the combustion chamber can take part in heat exchange during different steps of the enzymatic process.     

Refinement of the C222(1) crystal form of oxidized uteroglobin at 1.34 A resolution

The structure of uteroglobin, a progesterone binding protein from rabbit uterine fluid, was determined and refined at 1.34 A resolution to a conventional R-factor of 0.229. The accuracy of the co-ordinates is estimated to be 0.15 A. The isotropic temperature factor of individual atoms was refined and its average value is 11.9 A2 for the 548 non-hydrogen atoms of the protein monomer. A total of 83 water molecules was located in difference electron density maps and refined, first using a constant occupancy factor of 1 and then variable occupancy, the final (Q) being 0.63. The mean temperature factor of the water oxygen atoms is 26.4 A2. Uteroglobin is a dimer and its secondary structure consists of four alpha-helices per monomer that align in an anti-parallel fashion. There is one beta-turn between helix 2 and helix 3 (Lys26 to Glu29); 76% of the residues are part of the alpha-helices. In the core of the dimeric protein molecule, between the two monomers that are held together by two disulfide bridges, we have observed a closed cavity. Its length is 15.6 A and its width is 9 A; 14 water molecules could be positioned inside. In the "bottom" part of the protein, near the C terminus, we have observed a smaller cavity, occupied by two water molecules. The calculation of the molecular surface revealed four surface pockets whose possible functional implications are discussed below.     

Superoxide dismutase: fluctuations in the structure and solvation of the active site channel studied by molecular dynamics simulation

The molecular dynamics (MD) simulation of superoxide dismutase (SOD) in water is carried out for a total of 23 ps. The simulation system is a 26 A sphere centered at the active site of SOD, including 1602 atoms from SOD and 1761 water molecules. There is no gross deviation from the x-ray structure for the average MD structure. The structure and potential fluctuations around the active site are examined. The results provide new insight to the interactions between SOD and its substrate superoxide.     

Structure of native and apo carbonic anhydrase II and structure of some of its anion-ligand complexes.

In order to obtain a better structural framework for understanding the catalytic mechanism of carbonic anhydrase, a number of inhibitor complexes of the enzyme were investigated crystallographically. The three-dimensional structure of free human carbonic anhydrase II was refined at pH 7.8 (1.54 A resolution) and at pH 6.0 (1.67 A resolution). The structure around the zinc ion was identical at both pH values. The structure of the zinc-free enzyme was virtually identical with that of the native enzyme, apart from a water molecule that had moved 0.9 A to fill the space that would be occupied by the zinc ion. The complexes with the anionic inhibitors bisulfite and formate were also studied at neutral pH. Bisulfite binds with one of its oxygen atoms, presumably protonized, to the zinc ion and replaces the zinc water. Formate, lacking a hydroxyl group, is bound with its oxygen atoms not far away from the position of the non-protonized oxygen atoms of the bisulfite complex, i.e. at hydrogen bond distance from Thr199 N and at a position between the zinc ion and the hydrophobic part of the active site. The result of these and other studies have implications for our view of the catalytic function of the enzyme, since virtually all inhibitors share some features with substrate, product or expected transition states. A reaction scheme where electrophilic activation of carbon dioxide plays an important role in the hydration reaction is presented. In the reverse direction, the protonized oxygen of the bicarbonate is forced upon the zinc ion, thereby facilitating cleavage of the carbon-oxygen bond. This is achieved by the combined action of the anionic binding site, which binds carboxyl groups, the side-chain of threonine 199, which discriminates between hydrogen bond donors and acceptors, and hydrophobic interaction between substrate and the active site cavity. The required proton transfer between the zinc water and His64 can take place through water molecules 292 and 318.     

The mutability of enzyme active-site shape determinants

Investigations of enzyme action typically focus on elucidating the catalytic roles of hydrogen bonding interactions between polar active-site residues and substrate molecules. Less clear is the importance of non-hydrogen bonding contacts to enzymatic rate accelerations. To investigate the importance of such interactions in a model system, six residues that participate in van der Waals contacts with substrate glucose within the active site of Escherichia coli glucokinase were individually randomized via site-directed mutagenesis. In vivo selection in a glucokinase-deficient bacterium was employed to identify amino acid substitutions that were complicit with enzyme activity. The results suggest that small residues, such as alanine and glycine, are largely immutable, whereas larger amino acids are more tolerant of diverse substitution patterns. Surprisingly, a glucokinase variant that contains glycine in place of six non-hydrogen bonding contacts retains approximately 1% of the wild-type activity. These findings establish non-hydrogen bonding shape determinants as highly appealing targets for widespread substitution during efforts to redesign the catalytic properties of natural enzymes.     

X-ray structural analysis of magnesium-containing Serratia marcescens endonuclease]

The three-dimensional crystal structure of the DNA/RNA nonspecific endonuclease from Serratia marcescens was refined at the resolution of 1.07 A to R factor of 12.4% and Rfree factor of 15.3% using the anisotropic approximation. The structure includes 3924 non-hydrogen atoms, 715 protein-bound water molecules, and a Mg2+ ion in each binding site of each subunit of the nuclease homodimeric globular molecule. The 3D topological model of the enzyme was revealed, the inner symmetry of the monomers in its N- and C-termini was found, and the local environment of the magnesium cofactor in the nuclease active site was defined. Mg2+ ion was found to be bound to the Asn119 residue and surrounded by five associated water molecules that form an octahedral configuration. The coordination distances for the water molecules and the O delta 1 atom of Asn119 were shown to be within a range of 2.01-2.11 A. The thermal factors for the magnesium ion in subunits are 7.08 and 4.60 A2, and the average thermal factors for the surrounding water molecules are 11.14 and 10.30 A2, respectively. The region of the nuclease subunit interactions was localized, and the alternative side chain conformations were defined for 51 amino acid residues of the nuclease dimer.     

The three-dimensional structure of a glutathione S-transferase from the mu gene class. Structural analysis of the binary complex of isoenzyme 3-3 and glutathione at 2.2-A resolution.

The crystal structure of a mu class glutathione S-transferase (EC 2.5.1.18) from rat liver (isoenzyme 3-3) in complex with the physiological substrate glutathione (GSH) has been solved at 2.2-A resolution by multiple isomorphous replacement methods. The enzyme crystallized in the monoclinic space group C2 with unit cell dimensions of a = 87.98 A, b = 69.41 A, c = 81.34 A, and beta = 106.07 degrees. Oligonucleotide-directed site-specific mutagenesis played an important role in the solution of the structure in that the cysteine mutants C86S, C114S, and C173S were used to help locate the positions of mercuric ion sites in nonisomorphous derivatives with ethylmercuric phosphate and to align the sequence with the model derived from MIR phases. A complete model for the protein was not obtained until part of the solvent structure was interpreted. The dimer in the asymmetric unit refined to a crystallographic R = 0.171 for 19,298 data and I > or = 1.5 sigma (I). The final model consists of 4150 atoms, including all non-hydrogen atoms of 434 amino acid residues, two GSH molecules, and oxygen atoms of 474 water molecules. The dimeric enzyme is globular in shape with dimensions of 53 x 62 x 56 A. Crystal contacts are primarily responsible for conformational differences between the two subunits which are related by a noncrystallographic 2-fold axis. The structure of the type 3 subunit can be divided into two domains separated by a short linker, a smaller alpha/beta domain (domain I, residues 1-82), and a larger alpha domain (domain II, residues 90-217). Domain I contains four beta-strands which form a central mixed beta-sheet and three alpha-helices which are arranged in a beta alpha beta alpha beta beta alpha motif. Domain II is composed of five alpha-helices. Domain I can be considered the glutathione binding domain, while domain II seems to be primarily responsible for xenobiotic substrate binding. The active site is located in a deep (19-A) cavity which is composed of three relatively mobile structural elements: the long loop (residues 33-42) of domain I, the alpha 4/alpha 5 helix-turn-helix segment, and the C-terminal tail. GSH is bound at the active site in an extended conformation at one end of the beta-sheet of domain I with its backbone facing the cavity and the sulfur pointing toward the subunit to which it is bound.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crystal structure of Bacillus subtilis guanine deaminase: the first domain-swapped structure in the cytidine deaminase superfamily

Guanine deaminase, a key enzyme in the nucleotide metabolism, catalyzes the hydrolytic deamination of guanine into xanthine. The crystal structure of the 156-residue guanine deaminase from Bacillus subtilis has been solved at 1.17-A resolution. Unexpectedly, the C-terminal segment is swapped to form an intersubunit active site and an intertwined dimer with an extensive interface of 3900 A(2) per monomer. The essential zinc ion is ligated by a water molecule together with His(53), Cys(83), and Cys(86). A transition state analog was modeled into the active site cavity based on the tightly bound imidazole and water molecules, allowing identification of the conserved deamination mechanism and specific substrate recognition by Asp(114) and Tyr(156'). The closed conformation also reveals that substrate binding seals the active site entrance, which is controlled by the C-terminal tail. Therefore, the domain swapping has not only facilitated the dimerization but has also ensured specific substrate recognition. Finally, a detailed structural comparison of the cytidine deaminase superfamily illustrates the functional versatility of the divergent active sites found in the guanine, cytosine, and cytidine deaminases and suggests putative specific substrate-interacting residues for other members such as dCMP deaminases.     

Crystal structure of phosphatidylglycerophosphatase (PGPase), a putative membrane-bound lipid phosphatase, reveals a novel binuclear metal binding site and two "proton wires"

Phosphatidylglycerophosphatase (PGPase), an enzyme involved in lipid metabolism, catalyzes formation of phosphatidylglycerol from phosphatidylglycerophosphate. Phosphatidylglycerol is a multifunctional phospholipid, found in the biological membranes of many organisms. Here, we report the crystal structure of Listeria monocytogenes PGPase at 1.8 A resolution. PGPase, an all-helical molecule, forms a homotetramer. Each protomer contains an independent active site with two metal ions, Ca(2+) and Mg(2+), forming a hetero-binuclear center located in a hydrophilic cavity near the surface of the molecule. The binuclear center, conserved ligands, metal-bound water molecules, and an Asp-His dyad form the active site. The catalytic mechanism of this enzyme is likely to proceed via binuclear metal activated nucleophilic water. The binuclear metal-binding active-site environment of this structure should provide insights into substrate binding and metal-dependent catalysis. A long channel with inter-linked linear water chains, termed "proton wires," is observed at the tetramer interface. Comparison of similar water chain structures in photosynthetic reaction centers (RCs), Cytochrome f, gramicidin, and bacteriorhodopsin, suggests that PGPase may conduct protons via proton wires.     

A model of the pressure dependence of the enantioselectivity of Candida rugosalipase towards (+/-)-menthol

Transesterification of (+/-)-menthol using propionic acid anhydride and Candida rugosa lipase was performed in chloroform and water at different pressures (1, 10, 50, and 100 bar) to study the pressure dependence of enantioselectivity E. As a result, E significantly decreased with increasing pressure from E = 55 (1 bar) to E = 47 (10 bar), E = 37 (50 bar), and E = 9 (100 bar). To rationalize the experimental findings, molecular dynamics simulations of Candida rugosa lipase were carried out. Analyzing the lipase geometry at 1, 10, 50, and 100 bar revealed a cavity in the Candida rugosa lipase. The cavity leads from a position on the surface distinct from the substrate binding site to the core towards the active site, and is limited by F415 and the catalytic H449. In the crystal structure of the Candida rugosa lipase, this cavity is filled with six water molecules. The number of water molecules in this cavity gradually increased with increasing pressure: six molecules in the simulation at 1 bar, 10 molecules at 10 bar, 12 molecules at 50 bar, and 13 molecules at 100 bar. Likewise, the volume of the cavity progressively increased from about 1864 A(3) in the simulation at 1 bar to 2529 A(3) at 10 bar, 2526 A(3) at 50 bar, and 2617 A(3) at 100 bar. At 100 bar, one water molecule slipped between F415 and H449, displacing the catalytic histidine side chain and thus opening the cavity to form a continuous water channel. The rotation of the side chain leads to a decreased distance between the H449-N epsilon and the (+)-menthyl-oxygen (nonpreferred enantiomer) in the acyl enzyme intermediate, a factor determining the enantioselectivity of the lipase. Although the geometry of the preferred enantiomer is similar in all simulations, the geometry of the nonpreferred enantiomer gets gradually more reactive. This observation correlates with the gradually decreasing enantioselectivity E.     

Crystal structure of pyranose 2-oxidase from the white-rot fungus Peniophora sp

Pyranose 2-oxidase catalyzes the oxidation of a number of carbohydrates using dioxygen. The enzyme forms a D(2) symmetric homotetramer and contains one covalently bound FAD per subunit. The structure of the enzyme from Peniophora sp. was determined by multiwavelength anomalous diffraction (MAD) based on 96 selenium sites per crystallographic asymmetric unit and subsequently refined to good-quality indices. According to its chain fold, the enzyme belongs to the large glutathione reductase family and, in a more narrow sense, to the glucose-methanol-choline oxidoreductase (GMC) family. The tetramer contains a spacious central cavity from which the substrate enters one of the four active centers by penetrating a mobile barrier. Since this cavity can only be accessed by glucose-sized molecules, the enzyme does not convert sugars that are part of a larger molecule. The geometry of the active center and a comparison with an inhibitor complex of the homologous enzyme cellobiose dehydrogenase allow the modeling of the reaction at a high confidence level.     

Structure of the [2Fe-2S] ferredoxin I from the blue-green alga Aphanothece sacrum at 2.2 A resolution

Crystals of a [2Fe-2S] ferredoxin (Fd) I with a relative molecular mass of 10,480 were obtained from the blue-green alga Aphanothece sacrum. Each asymmetric unit of the crystal contains four molecules. An electron density map calculated by the single isomorphous replacement method with the anomalous dispersion at 2.5 A resolution was refined by averaging the four molecules in the asymmetric unit. Positional and isotropic thermal parameters for the non-hydrogen atoms of the four molecules and 158 water molecules were refined to an R-factor (R = sigma[Fo-Fc[/sigma Fo) of 0.23 by the restrained least-squares method. The estimated root-mean-square (r.m.s.) error for the atomic positions is 0.3 A. The r.m.s. deviations of equivalent C alpha atoms of the asymmetric-unit molecules superposed by the least-squares method average 0.35 A. The Fd molecule has a structure like the beta-barrel in the molecule of the [2Fe-2S] Fd from Spirulina platensis. A [2Fe-2S] cluster is bonded covalently to the protein molecule by four Fe-S, in which three of the Fe-S bonds are in a loop segment from position 38 to 47. The hydrophobic core inside the beta-barrel is formed by seven conservative residues: Val15, Val18, Ile24, Leu51, Ile74, Ala79 and Ile87. The molecular surface around Tyr23, Tyr80 and the active center may interact with ferredoxin-NADP+ reductase. One of the two iron atoms of the [2Fe-2S] cluster should be more easily reduced than the other because of differences in the hydrogen-bonding scheme and the hydrophobicity around the atoms.     

Hydration energy landscape of the active site cavity in cytochrome P450cam

Hydration of protein cavities influences protein stability, dynamics, and function. Protein active sites usually contain water molecules that, upon ligand binding, are either displaced into bulk solvent or retained to mediate protein–ligand interactions. The contribution of water molecules to ligand binding must be accounted for to compute accurate values of binding affinities. This requires estimation of the extent of hydration of the binding site. However, it is often difficult to identify the water molecules involved in the binding process when ligands bind on the surface of a protein. Cytochrome P450cam is, therefore, an ideal model system because its substrate binds in a buried active site, displacing partially disordered solvent, and the protein is well characterized experimentally. We calculated the free energy differences for having five to eight water molecules in the active site cavity of the unliganded enzyme from molecular dynamics simulations by thermodynamic integration employing a three-stage perturbation scheme. The computed free energy differences between the hydration states are small (within 12 kJ mol⁻¹) but distinct. Consistent with the crystallographic determination and studies employing hydrostatic pressure, we calculated that, although ten water molecules could in principle occupy the volume of the active site, occupation by five to six water molecules is thermodynamically most favorable. Proteins 32:381–396, 1998. © 1998 Wiley-Liss, Inc.     

The structure of human microsomal cytochrome P450 3A4 determined by X-ray crystallography to 2.05-A resolution

The structure of P450 3A4 was determined by x-ray crystallography to 2.05-A resolution. P450 3A4 catalyzes the metabolic clearance of a large number of clinically used drugs, and a number of adverse drug-drug interactions reflect the inhibition or induction of the enzyme. P450 3A4 exhibits a relatively large substrate-binding cavity that is consistent with its capacity to oxidize bulky substrates such as cyclosporin, statins, taxanes, and macrolide antibiotics. Family 3A P450s also exhibit unusual kinetic characteristics that suggest simultaneous occupancy by smaller substrates. Although the active site volume is similar to that of P450 2C8 (PDB code: 1PQ2), the shape of the active site cavity differs considerably due to differences in the folding and packing of portions of the protein that form the cavity. Compared with P450 2C8, the active site cavity of 3A4 is much larger near the heme iron. The lower constraints on the motions of small substrates near the site of oxygen activation may diminish the efficiency of substrate oxidation, which may, in turn, be improved by space restrictions imposed by the presence of a second substrate molecule. The structure of P450 3A4 should facilitate a better understanding of the substrate selectivity of the enzyme.     

The crystal structure of a novel,inactive, lysine 49 PLA2 from Agkistrodon acutus venom: an ultrahigh resolution,AB initio structure determination

The crystal structure of acutohaemolysin, a lysine 49 phospholipase A2 protein with 1010 non-hydrogen protein atoms and 232 water molecules, has been determined ab initio using the program SnB at an ultrahigh resolution of 0.8 A. The lack of catalytic activity appears to be related to the presence of Phe102, which prevents the access of substrate to the active site. The substitution of tryptophan for leucine at residue 10 interferes with dimer formation and may be responsible for the additional loss of hemolytic activity. The ultrahigh resolution of the experimental diffraction data permits alternative conformations to be modeled for disordered residues, many hydrogen atoms to be located, the protonation of the Nepsilon2 atom in the catalytic residue His48 to be observed experimentally, and the density of the bonding electrons to be analyzed in detail.     

Structure of cobalt carbonic anhydrase complexed with bicarbonate.

The three-dimensional structure of a complex between catalytically active cobalt(II) substituted human carbonic anhydrase II and its substrate bicarbonate was determined by X-ray crystallography (1.9 A). One water molecule and two bicarbonate oxygen atoms are found at distances between 2.3 and 2.5 A from the cobalt ion in addition to the three histidyl ligands contributed by the peptide chain. The tetrahedral geometry around the metal ion in the native enzyme with a single water molecule 2.0 A from the metal is therefore lost. The geometry is difficult to classify but might best be described as distorted octahedral. The structure is suggested to represent a water-bicarbonate exchange state relevant also for native carbonic anhydrase, where the two unprotonized oxygen atoms of the substrate are bound in a carboxylate binding site and the hydroxyl group is free to move closer to the metal thereby replacing the metal-bound water molecule. A reaction mechanism based on crystallographically determined enzyme-ligand complexes is represented.     

Enhancement of catalytic efficiency of enzymes through exposure to anhydrous organic solvent at 70 degrees C. Three-dimensional structure of a treated serine proteinase at 2.2 A resolution

The enzyme behavior in anhydrous media has important applications in biotechnology. So far chemical modifications and protein engineering have been used to alter the catalytic power of the enzymes. For the first time, it is demonstrated that an exposure of enzyme to anhydrous organic solvents at optimized high temperature enhances its catalytic power through local changes at the binding region. Six enzymes: proteinase K, wheat germ acid phosphatase, alpha-amylase, beta-glucosidase, chymotrypsin and trypsin have been exposed to acetonitrile at 70 degrees C for three hours. The activities of these enzymes were found to be considerably enhanced. In order to understand the basis of this change in the activity of these enzymes, the structure of one of these treated enzymes, proteinase K has been analyzed in detail using X-ray diffraction method. The overall structure of the enzyme is similar to the native structure in aqueous environment. The hydrogen bonding system of the catalytic triad is intact after the treatment. However, the water structure in the substrate binding site undergoes some rearrangement as some of the water molecules are either displaced or completely absent. The most striking observation concerning the water structure pertains to the complete deletion of the water molecule which occupied the position at the so-called oxyanion hole in the active site of the native enzyme. Three acetonitrile molecules were found in the present structure. All the acetonitrile molecules are located in the recognition site. The sites occupied by acetonitrile molecules are independent of water molecules. The acetonitrile molecules are involved in extensive interactions with the protein atoms. All of them are interlinked through water molecules. The methyl group of one of the acetonitrile molecules (CCN1) interacts simultaneously with the hydrophobic side chains of Leu-96, Ile-107, and Leu-133. The development of such a hydrophobic environment at the recognition site introduces a striking conformation change in Ile-107 by rotating its side chain about C(alpha)--C(beta) bond by 180 degrees to bring about the delta-methyl group within the range of attractive van der Waals interactions with the methyl group of CCN1. A similar change has earlier been observed in proteinase K when it is complexed to a substrate analog lactoferrin fragment.     

Staphylococcus aureus aminopeptidase S is a founding member of a new peptidase clan

Staphylococcus aureus aminopeptidase S (AmpS) has been named for its predicted, but experimentally untested, aminopeptidase activity. The enzyme is homologous to biochemically characterized aminopeptidases that contain two cobalt or zinc ions in their active centers, but it is unrelated to all structurally characterized metallopeptidases. Here, we demonstrate AmpS aminopeptidase activity experimentally, and we present the 1.8-A crystal structure of the enzyme. Two metal ions with full occupancy and a third metal ion with low occupancy are present in the active site. A water molecule and Glu-319 serve as bridging ligands to the two metals with full occupancy. One of these metal ions is additionally coordinated by Glu-253 and His-348 and the other by His-381 and Asp-383. In addition, the metals are involved in weak metal-donor interactions to a water molecule and to Tyr-355. In the crystal, AmpS forms a dimer with a large internal cavity. The active sites are located at opposite ends of this internal cavity and are essentially inaccessible from the outside, suggesting that an inactive conformation was crystallized. Because gel filtration and analytical ultracentrifugation data also suggest dimer formation, the problem of substrate access to the active site cavity remains unresolved.     

Enhancement of catalytic activity of enzymes by heating in anhydrous organic solvents: 3D structure of a modified serine proteinase at high resolution

For the first time, it is demonstrated that exposure of an enzyme to anhydrous organic solvents at optimized high temperature enhances its catalytic power through local changes at the binding region. Six enzymes, namely, proteinase K, wheat germ acid phosphatase, alpha-amylase, beta-glucosidase, chymotrypsin and trypsin were exposed to acetonitrile at 70 degrees C for three hr. The activities of these enzymes were found to be considerably enhanced. In order to understand the basis of this change in the activity of these enzymes, proteinase K was analyzed in detail using X-ray diffraction method. The overall structure of the enzyme was found to be similar to the native structure in aqueous environment. The hydrogen bonding system of the catalytic triad remained intact after the treatment. However, the water structure in the substrate binding site underwent some rearrangement as some of the water molecules were either displaced or completely absent. The most striking observation concerning the water structure was the complete deletion of the water molecule which occupied the position at the so-called oxyanion hole in the active site of the native enzyme. Three acetonitrile molecules were found in the present structure. All the acetonitrile molecules were located in the recognition site. Interlinked through water molecules, the sites occupied by acetonitrile molecules were independent of water molecules. The acetonitrile molecules are involved in extensive interactions with the protein atoms. The methyl group of one of the acetonitrile molecules (CCN1) interacts simultaneously with the hydrophobic side chains of Leu 96, Ile 107 and Leu 133. The development of such a hydrophobic environment at the recognition site introduced a striking conformation change in Ile 107 by rotating its side chain about C alpha-C beta bond by 180 degrees to bring about the delta-methyl group within the range of attractive van der Waals interactions with the methyl group of CCN1. A similar change had earlier been observed in proteinase K when it was complexed to a substrate analogue, lactoferrin fragment.