Structure of the Complex of Bovine Pancreatic Phospholipase A2 with a Transition-State Analogue

1998 ◽  
Vol 54 (3) ◽  
pp. 334-341 ◽  
Author(s):  
Kanagaraj Sekar ◽  
Amarendra Kumar ◽  
Xiaohong Liu ◽  
Ming-Daw Tsai ◽  
Michael H. Gelb ◽  
...  
Keyword(s):  
Hydrogen Bonds ◽  
Transition State ◽  
Active Site ◽  
Calcium Ion ◽  
Hydroxyl Group ◽  
Pentagonal Bipyramid ◽  
Water Molecules ◽  
Phosphonate Group ◽  
Pancreatic Phospholipase ◽  
Transition State Analogue

The 1.89 Å resolution structure of the complex of bovine pancreatic phospholipase A2 (PLA2) with the transition-state analogue L-1-O-octyl-2-heptylphosphonyl-sn-glycero-3-phosphoethanolamine (TSA) has been determined. The crystal of the complex is trigonal, space group P3121, a = b = 46.58 and c = 102.91 Å and isomorphous to the native recombinant wild type (WT). The structure was refined to a final crystallographic R value of 18.0% including 957 protein atoms, 88 water molecules, one calcium ion and all 31 non-H atoms of the inhibitor at 1.89 Å resolution. In all, 7 726 reflections [F\gt2\sigma(F)] were used between 8.0 and 1.89 Å resolution. The inhibitor is deeply locked into the active-site cleft and coordinates to the calcium ion by displacing the two water molecules in the calcium pentagonal bipyramid by the anionic O atoms of the phosphate and phosphonate group. The hydroxyl group of Tyr69 hydrogen bonds to the second anionic O atom of the phosphate group while that of the phosphonate group replaces the third water, `catalytic' water, which forms a hydrogen bond to Nδ1 of His48. The fourth water which also shares Nδ1 of His48 is displaced by the steric hinderance of the inhibitor. The fifth conserved structural water is still present in the active site and forms a network of hydrogen bonds with the surrounding residues. The structure is compared with the other known TSA–PLA2 complexes.

scholarly journals A Trojan horse transition state analogue generated by MgF3- formation in an enzyme active site

2006 ◽  
Vol 103 (40) ◽  
pp. 14732-14737 ◽  
Author(s):  
N. J. Baxter ◽  
L. F. Olguin ◽  
M. Golicnik ◽  
G. Feng ◽  
A. M. Hounslow ◽  
...  
Keyword(s):  
Transition State ◽  
Active Site ◽  
Trojan Horse ◽  
Enzyme Active Site ◽  
Transition State Analogue

Structures of the catalytic site mutants D99A and H48Q and the calcium-loop mutant D49E of phospholipase A2

1999 ◽  
Vol 55 (2) ◽  
pp. 443-447 ◽  
Author(s):  
K. Sekar ◽  
R. Biswas ◽  
Y. Li ◽  
M.-D. Tsai ◽  
M. Sundaralingam
Keyword(s):  
Active Site ◽  
Recombinant Enzyme ◽  
Pentagonal Bipyramid ◽  
Water Molecules ◽  
Carboxylate Group ◽  
Structural Water ◽  
Space Group ◽  
Structural Perturbations ◽  
Active Site Mutants ◽  
Bonding Scheme

Crystal structures of the active-site mutants D99A and H48Q and the calcium-loop mutant D49E of bovine phospholipase A2 have been determined at around 1.9 Å resolution. The D99A mutant is isomorphous to the orthorhombic recombinant enzyme, space group P212121. The H48Q and the calcium-loop mutant D49E are isomorphous to the trigonal recombinant enzyme, space group P3121. The two active-site mutants show no major structural perturbations. The structural water is absent in D99A and, therefore, the hydrogen-bonding scheme is changed. In H48Q, the catalytic water is present and hydrogen bonded to Gln48 N, but the second water found in native His48 is absent. In the calcium-loop mutant D49E, the two water molecules forming the pentagonal bipyramid around calcium are absent and only one O atom of the Glu49 carboxylate group is coordinated to calcium, resulting in only four ligands.

Hydrophobic `lock and key' recognition of N-4-nitrobenzoylamino acid by strychnine

2006 ◽  
Vol 62 (6) ◽  
pp. 1061-1070 ◽  
Author(s):  
Agata Białońska ◽  
Zbigniew Ciunik
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bonds ◽  
Self Assembly ◽  
Chiral Discrimination ◽  
Hydroxyl Group ◽  
Water Molecules ◽  
Common Features ◽  
Protonated Amine ◽  
Hydrogen Bond Networks ◽  
Solvent Molecules

During racemic resolution of N-4-nitrobenzoyl-DL-amino acids (alanine, serine and aspartic acid) by a fractional crystallization of strychninium salts, crystals of both diastereomeric salts were grown, and the crystal structures of strychninium N-4-nitrobenzoyl-L-alaninate methanol disolvate (1a), strychninium N-4-nitrobenzoyl-D-alaninate dihydrate (1b), strychninium N-4-nitrobenzoyl-D-serinate dihydrate (2a), strychninium N-4-nitrobenzoyl-L-serinate methanol solvate hydrate (2b), strychninium hydrogen N-4-nitrobenzoyl-L-aspartate 3.75 hydrate (3a) and strychninium hydrogen N-4-nitrobenzoyl-D-aspartate 2.25 hydrate (3b) were determined. The strychninium cations form corrugated layers, which are separated by hydrogen-bonded anions and solvent molecules. Common features of the corrugated layers are deep hydrophobic grooves at their surfaces, which are occupied by the 4-nitrobenzoyl groups of suitable anions. The hydrophobic `lock and key' recognition of 4-nitrobenzoyl groups of amino acid derivatives in deep grooves of the strychnine self-assembly causes the resulting surface to have more hydrophilic properties, which are more appropriate for interactions in the hydrophilic environments from which strychninium salts were crystallized. In the crystal structure of (2a) and (3a), such hydrophobic `lock and key' recognition is responsible for the lack of N—H+...O− hydrogen bonds that are usually formed between the protonated tertiary amine N atom of the strychninium cation and the deprotonated carboxyl group of the resolved acid. In the crystal structure of (2a) and (3a), the protonated amine N atom is a donor of hydrogen bonds, while the hydroxyl group of the serine derivative and water molecules are their acceptors. In light of the hydrophobic recognition, chiral discrimination depends on the nature of the hydrogen-bond networks, which involve anions, solvent molecules and the protonated amine N atom of strychninium cations.

1.72 Å Resolution Refinement of the Trigonal Form of Bovine Pancreatic Phospholipase A2

1998 ◽  
Vol 54 (3) ◽  
pp. 342-346 ◽  
Author(s):  
K. Sekar ◽  
C. Sekharudu ◽  
M.-D. Tsai ◽  
M. Sundaralingam
Keyword(s):  
Active Site ◽  
Water Molecules ◽  
Unit Cell Parameters ◽  
Structural Water ◽  
Cell Parameters ◽  
Pancreatic Phospholipase ◽  
Pancreatic Phospholipase A2 ◽  
Solvent Molecules ◽  
Hydrolysis Of ◽  
The Ideal

The trigonal crystal structure of the recombinant bovine pancreatic phospholipase A2 has been re-refined at a slightly higher resolution (1.72 Å). The crystals are trigonal, space group P3121, unit-cell parameters a = b = 46.78 and c = 102.89 Å and are isomorphous to the previous structure. The structure was refined to a final crystallographic R value of 19.5% (R free = 28.4%) using 10 531 reflections. A total of 106 solvent molecules were included in the refinement compared with the earlier refinement which contains only 85 water molecules and 8 925 reflections at 1.8 Å resolution. The root-mean-square deviation from the ideal bond lengths and bond angles is considerably better in the present refinement. The active site is extended (\sim14 Å) from Ala1 to the calcium. The three catalytic residues (Asp99, His48 and the catalytic water) are connected by the conserved structural water and the N-terminal Ala1 on one side, and by the calcium through an equatorial water on the other. The water molecules play a role in the activity of the enzyme PLA2. The Ala1 end of the extended active site performs the activation of the phospholipid membranes while the opposite end performs the hydrolysis of the monomeric phospholipids.

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