PDBsum entry 1ceh

Hydrolase (carboxylic ester)

PDB id: 1ceh

Contents

Protein chain

123 a.a.

Metals

_CA

Waters ×81

References listed in PDB file

Key reference

Title

Structure and function of the catalytic site mutant asp 99 asn of phospholipase a2: absence of the conserved structural water.

Authors

A.Kumar, C.Sekharudu, B.Ramakrishnan, C.M.Dupureur, H.Zhu, M.D.Tsai, M.Sundaralingam.

Ref.

Protein Sci, 1994, 3, 2082-2088. [DOI no: 10.1002/pro.5560031121]

PubMed id

7703854

Note In the PDB file this reference is annotated as "TO BE PUBLISHED". The citation details given above were identified by an automated search of PubMed on title and author names, giving a percentage match of 86%.

Abstract

To probe the role of the Asp-99 ... His-48 pair in phospholipase A2 (PLA2) catalysis, the X-ray structure and kinetic characterization of the mutant Asp-99-->Asn-99 (D99N) of bovine pancreatic PLA2 was undertaken. Crystals of D99N belong to the trigonal space group P3(1)21 and were isomorphous to the wild type (WT) (Noel JP et al., 1991, Biochemistry 30:11801-11811). The 1.9-A X-ray structure of the mutant showed that the carbonyl group of Asn-99 side chain is hydrogen bonded to His-48 in the same way as that of Asp-99 in the WT, thus retaining the tautomeric form of His-48 and the function of the enzyme. The NH2 group of Asn-99 points away from His-48. In contrast, in the D102N mutant of the protease enzyme trypsin, the NH2 group of Asn-102 is hydrogen bonded to His-57 resulting in the inactive tautomeric form and hence the loss of enzymatic activity. Although the geometry of the catalytic triad in the PLA2 mutant remains the same as in the WT, we were surprised that the conserved structural water, linking the catalytic site with the ammonium group of Ala-1 of the interfacial site, was ejected by the proximity of the NH2 group of Asn-99. The NH2 group now forms a direct hydrogen bond with the carbonyl group of Ala-1.

Figure 3.
Fig. 3. A: Hypothtical hydrogen bonding scheme for the D99N mutant with theNH2 group of Asn-99 hydrogen bonding to His- 48 and Tyr-52. B The alternateconformationofthe Asn-99 side chain with the NH group pointing away from the His-48 nd hy- drogen bonding to Tyr-73 and the structural water-3 molecule. The hydroxyl groups of Tyr-52 nd Tyr-73 have switched orientation from A to B to accommodatethe hydrogen bonding. Notice hat the hydrogen bonding pattern of thestructural water molecule in A and B is different from each other and the WT lthough the structural water is 4-coordinated in all3 cases. C: The hydrogen bonding scheme in the X-ray structure of themutant D99N. The structural water is missing andthe mino roup Asn-99 is directly hydrogen bonded to thecarbonl group of Ala-1.

Figure 4.
Fig. 4. Van der Waals surface diagram ofthe residues aroundthe structural water in the WT (A) an the D99N mutant (B). Notice that the new position and conformation of la-1 in B fiis the void created by the missing structural water. C: Overlay fthe skele- tal drawings ofthe WT (red) and hemtant D99N (green) show- ing the hydrogen bonding scheme. Residues around Ala-I that are hydrogen bonded to theammoniumgroup are also Notice the large change in theorientationofthe NH3+and p groups of Ala-1. Despite this change, theammonium group in the mutant still forms 3 hydrogen bonds, 2 f which areto thesame acceptorsas in he WT.

The above figures are reprinted from an Open Access publication published by the Protein Society: Protein Sci (1994, 3, 2082-2088) copyright 1994.

Secondary reference #1

Title

Phospholipase a2 engineering. X-Ray structural and functional evidence for the interaction of lysine-56 with substrates.

Authors

J.P.Noel, C.A.Bingman, T.L.Deng, C.M.Dupureur, K.J.Hamilton, R.T.Jiang, J.G.Kwak, C.Sekharudu, M.Sundaralingam, M.D.Tsai.

Ref.

Biochemistry, 1991, 30, 11801-11811. [DOI no: 10.1021/bi00115a010]

PubMed id

1751497