Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K.


Abstract

The catalytic contribution of His48 in the active site of porcine pancreatic phospholipase A2 was examined using site-directed mutagenesis. Replacement of His48 by lysine (H48K) gives rise to a protein having a distorted lipid binding pocket. Activity of this variant drops below the detection limit which is 10(7)-fold lower than that of the wild-type enzyme. On the other hand, the presence of glutamine (H48Q) or asparagine (H48N) at this position does not affect the structural integrity of the enzyme as can be derived from the preserved lipid binding properties of these variants. However, the substitutions H48Q and H48N strongly reduce the turnover number, i.e. by a factor of 10(5). Residual activity is totally lost after addition of a competitive inhibitor. We conclude that proper lipid binding on its own accelerates ester bond hydrolysis by a factor of 10(2). With the selected variants, we were also able to dissect the contribution of the hydrogen bond between Asp99 and His48 on conformational stability, being 5.2 kJ/mol. Another hydrogen bond with His48 is formed when the competitive inhibitor (R)-2-dodecanoylamino-hexanol-1-phosphoglycol interacts with the enzyme. Its contribution to binding of the inhibitor in the presence of an interface was found to be 5.7 kJ/mol. Study holds ProTherm entries: 5540, 5541, 5542, 5543 Extra Details: additive : EDTA(2 mM),

Submission Details

ID: U9jSTRr43

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:30 p.m.

Version: 1

Publication Details
Janssen MJ;van de Wiel WA;Beiboer SH;van Kampen MD;Verheij HM;Slotboom AJ;Egmond MR,Protein Eng. (1999) Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K. PMID:10388847
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
1FX9 2000-09-25T00:00:00+0000 2.0 CARBOXYLIC ESTER HYDROLASE COMPLEX (DIMERIC PLA2 + MJ33 INHIBITOR + SULPHATE IONS)
1FXF 2000-09-25T00:00:00+0000 1.85 CARBOXYLIC ESTER HYDROLASE COMPLEX (DIMERIC PLA2 + MJ33 INHIBITOR + PHOSPHATE IONS)
1HN4 2000-12-06T00:00:00+0000 1.5 PROPHOSPHOLIPASE A2 DIMER COMPLEXED WITH MJ33, SULFATE, AND CALCIUM
1L8S 2002-03-21T00:00:00+0000 1.55 CARBOXYLIC ESTER HYDROLASE COMPLEX (DIMERIC PLA2 + LPC-ether + ACETATE + PHOSPHATE IONS)
1P2P 1983-06-27T00:00:00+0000 2.6 STRUCTURE OF PORCINE PANCREATIC PHOSPHOLIPASE A2 AT 2.6 ANGSTROMS RESOLUTION AND COMPARISON WITH BOVINE PHOSPHOLIPASE A2
1PIR 1994-12-22T00:00:00+0000 0 SOLUTION STRUCTURE OF PORCINE PANCREATIC PHOSPHOLIPASE A2
1PIS 1994-12-22T00:00:00+0000 0 SOLUTION STRUCTURE OF PORCINE PANCREATIC PHOSPHOLIPASE A2
1SFV 1996-02-20T00:00:00+0000 0 PORCINE PANCREAS PHOSPHOLIPASE A2, NMR, MINIMIZED AVERAGE STRUCTURE
1SFW 1996-02-23T00:00:00+0000 0 PORCINE PANCREAS PHOSPHOLIPASE A2, NMR, 18 STRUCTURES
1Y6O 2004-12-06T00:00:00+0000 2.0 Crystal structure of disulfide engineered porcine pancreatic phospholipase A2 to group-X isozyme in complex with inhibitor MJ33 and phosphate ions

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
96.8 Phospholipase A2, major isoenzyme P04416 PA22_PIG
100.0 Phospholipase A2, major isoenzyme P00592 PA21B_PIG