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
1BVM 1999-09-16 SOLUTION NMR STRUCTURE OF BOVINE PANCREATIC PHOSPHOLIPASE A2, 20 STRUCTURES
1G4I 2001-04-04 0.97 Crystal structure of the bovine pancreatic phospholipase A2 at 0.97A
1VL9 2004-10-19 0.97 Atomic resolution (0.97A) structure of the triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2
2BCH 2006-07-04 1.1 A possible of Second calcium ion in interfacial binding: Atomic and Medium resolution crystal structures of the quadruple mutant of phospholipase A2
2BAX 2005-10-25 1.1 Atomic Resolution Structure of the Double Mutant (K53,56M) of Bovine Pancreatic Phospholipase A2
1UNE 1998-05-06 1.5 CARBOXYLIC ESTER HYDROLASE, 1.5 ANGSTROM ORTHORHOMBIC FORM OF THE BOVINE RECOMBINANT PLA2
1VKQ 2004-08-31 1.6 A re-determination of the structure of the triple mutant (K53,56,120M) of phospholipase A2 at 1.6A resolution using sulphur-SAS at 1.54A wavelength
4BP2 1991-10-15 1.6 CRYSTALLOGRAPHIC REFINEMENT OF BOVINE PRO-PHOSPHOLIPASE A2 AT 1.6 ANGSTROMS RESOLUTION
2B96 2006-03-28 1.7 Third Calcium ion found in an inhibitor bound phospholipase A2
1BP2 1981-05-21 1.7 STRUCTURE OF BOVINE PANCREATIC PHOSPHOLIPASE A2 AT 1.7 ANGSTROMS RESOLUTION
1MKT 1998-03-11 1.72 CARBOXYLIC ESTER HYDROLASE, 1.72 ANGSTROM TRIGONAL FORM OF THE BOVINE RECOMBINANT PLA2 ENZYME
1BPQ 1993-10-31 1.8 PHOSPHOLIPASE A2 ENGINEERING. X-RAY STRUCTURAL AND FUNCTIONAL EVIDENCE FOR THE INTERACTION OF LYSINE-56 WITH SUBSTRATES
1MKU 1997-12-24 1.8 CARBOXYLIC ESTER HYDROLASE, ORTHORHOMBIC FORM OF THE TRIPLE MUTANT
2BPP 1993-10-31 1.8 PHOSPHOLIPASE A2 ENGINEERING. X-RAY STRUCTURAL AND FUNCTIONAL EVIDENCE FOR THE INTERACTION OF LYSINE-56 WITH SUBSTRATES
1O3W 2003-07-15 1.85 Structure of the inhibitor free triple mutant (K53,56,120M) of phospholipase A2
1MKV 1998-03-18 1.89 CARBOXYLIC ESTER HYDROLASE COMPLEX (PLA2 + TRANSITION STATE ANALOG COMPLEX)
1CEH 1995-02-07 1.9 STRUCTURE AND FUNCTION OF THE CATALYTIC SITE MUTANT ASP99ASN OF PHOSPHOLIPASE A2: ABSENCE OF CONSERVED STRUCTURAL WATER
1C74 2000-07-22 1.9 Structure of the double mutant (K53,56M) of phospholipase A2
1GH4 2001-05-09 1.9 Structure of the triple mutant (K56M, K120M, K121M) of phospholipase A2
1IRB 1997-12-24 1.9 CARBOXYLIC ESTER HYDROLASE
1KVY 1998-11-18 1.9 CARBOXYLIC ESTER HYDROLASE, SINGLE MUTANT D49E COORDINATED TO CALCIUM
2ZP3 2008-11-04 1.9 Carboxylic ester hydrolase, single mutant d49n of bovine pancreatic pla2 enzyme
1KVX 1998-11-18 1.9 CARBOXYLIC ESTER HYDROLASE, SINGLE MUTANT D99A OF BOVINE PANCREATIC PLA2, 1.9 A ORTHORHOMBIC FORM
2ZP5 2008-11-04 1.9 Carboxylic ester hydrolase, single mutant d49k of bovine pancreatic pla2 enzyme
2BD1 2006-07-04 1.9 A possible role of the second calcium ion in interfacial binding: Atomic and medium resolution crystal structures of the quadruple mutant of phospholipase A2
1MKS 1997-12-24 1.9 CARBOXYLIC ESTER HYDROLASE, TRIGONAL FORM OF THE TRIPLE MUTANT
2ZP4 2008-11-04 1.9 Carboxylic ester hydrolase, single mutant h48n of bovine pancreatic pla2 enzyme
1FDK 1998-10-14 1.91 CARBOXYLIC ESTER HYDROLASE (PLA2-MJ33 INHIBITOR COMPLEX)
1KVW 1998-11-18 1.95 CARBOXYLIC ESTER HYDROLASE, SINGLE MUTANT H48Q OF BOVINE PANCREATIC PLA2 ENZYME
3BP2 1983-09-15 2.1 ROLE OF THE N-TERMINUS IN THE INTERACTION OF PANCREATIC PHOSPHOLIPASE A2 WITH AGGREGATED SUBSTRATES. PROPERTIES AND CRYSTAL STRUCTURE OF TRANSAMINATED PHOSPHOLIPASE A2
1O2E 2003-09-09 2.6 Structure of the triple mutant (K53,56,120M) + Anisic acid complex of phospholipase A2
2BP2 1981-07-16 3.0 THE STRUCTURE OF BOVINE PANCREATIC PROPHOSPHOLIPASE A2 AT 3.0 ANGSTROMS RESOLUTION

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