Phospholipase A2 engineering. The structural and functional roles of aromaticity and hydrophobicity in the conserved phenylalanine-22 and phenylalanine-106 aromatic sandwich.


Abstract

The highly conserved phenylalanine-22 and phenylalanine-106, arranged as an aromatic sandwich, form part of an invariant hydrophobic wall that shields the active site of bovine pancreatic phospholipase A2 (PLA2) from bulk solvent [Dijkstra, B. W., Drenth, J., & Kalk, K. H. (1981) Nature 289, 604-606]. The residues have also been suggested to interact with the sn-2 acyl chain of bound phospholipid substrate [White, S. P., Scott, D. L., Otwinowski, Z., Gelb, M. H., & Sigler, P. B. (1990) Science 250, 1560-1563]. We now report the importance of these two residues in the structure and function of PLA2 in terms of aromaticity (changing to Ile) and hydrophobic (changing to Ala) and hydrophilic (changing to Tyr) character of these residues. The structural properties of the mutants were analyzed by proton NMR and by guanidine hydrochloride-induced denaturation. The functional properties were determined by measuring kinetic parameters toward various substrates in the forms of monomers, micelles, and vesicles, and by measuring equilibrium dissociation constants at the interface. The results show that (i) The conformational stability of each mutant was as good as that of wild-type PLA2; none of the mutants was significantly perturbed structurally as judged from detailed 1H NMR analysis. These results suggest that neither the Phe-22/Phe-106 face-to-face pair nor the Phe-22/Tyr-111 edge-to-face pair plays a significant structural role. (ii) Mutations to Ile at either position 22 or position 106 resulted in only minor perturbations in activity. This suggests that the aromaticity is not important to the function of these two residues.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 502, 503, 504, 505, 506, 507, 508 Extra Details: Phospholipase A2; PLA2; hydrophobicity; conformational stability;,activity

Submission Details

ID: 72RcyNWU

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:15 p.m.

Version: 1

Publication Details
Dupureur CM;Yu BZ;Mamone JA;Jain MK;Tsai MD,Biochemistry (1992) Phospholipase A2 engineering. The structural and functional roles of aromaticity and hydrophobicity in the conserved phenylalanine-22 and phenylalanine-106 aromatic sandwich. PMID:1420172
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1BP2 1981-04-06T00:00:00+0000 1.7 STRUCTURE OF BOVINE PANCREATIC PHOSPHOLIPASE A2 AT 1.7 ANGSTROMS RESOLUTION
1BPQ 1991-10-28T00:00:00+0000 1.8 PHOSPHOLIPASE A2 ENGINEERING. X-RAY STRUCTURAL AND FUNCTIONAL EVIDENCE FOR THE INTERACTION OF LYSINE-56 WITH SUBSTRATES
1BVM 1998-09-14T00:00:00+0000 0 SOLUTION NMR STRUCTURE OF BOVINE PANCREATIC PHOSPHOLIPASE A2, 20 STRUCTURES
1C74 2000-01-22T00:00:00+0000 1.9 Structure of the double mutant (K53,56M) of phospholipase A2
1CEH 1994-11-16T00:00:00+0000 1.9 STRUCTURE AND FUNCTION OF THE CATALYTIC SITE MUTANT ASP99ASN OF PHOSPHOLIPASE A2: ABSENCE OF CONSERVED STRUCTURAL WATER
1FDK 1997-09-04T00:00:00+0000 1.91 CARBOXYLIC ESTER HYDROLASE (PLA2-MJ33 INHIBITOR COMPLEX)
1G4I 2000-10-27T00:00:00+0000 0.97 Crystal structure of the bovine pancreatic phospholipase A2 at 0.97A
1GH4 2000-11-09T00:00:00+0000 1.9 Structure of the triple mutant (K56M, K120M, K121M) of phospholipase A2
1IRB 1997-08-13T00:00:00+0000 1.9 CARBOXYLIC ESTER HYDROLASE
1KVW 1998-04-24T00:00:00+0000 1.95 CARBOXYLIC ESTER HYDROLASE, SINGLE MUTANT H48Q OF BOVINE PANCREATIC PLA2 ENZYME

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
95.1 Phospholipase A2 P14419 PA21B_SHEEP
100.0 Phospholipase A2 P00593 PA21B_BOVIN