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
1SFW 1996-07-11 PORCINE PANCREAS PHOSPHOLIPASE A2, NMR, 18 STRUCTURES
1PIS 1995-06-03 SOLUTION STRUCTURE OF PORCINE PANCREATIC PHOSPHOLIPASE A2
1PIR 1995-06-03 SOLUTION STRUCTURE OF PORCINE PANCREATIC PHOSPHOLIPASE A2
1SFV 1996-07-11 PORCINE PANCREAS PHOSPHOLIPASE A2, NMR, MINIMIZED AVERAGE STRUCTURE
1HN4 2001-12-05 1.5 PROPHOSPHOLIPASE A2 DIMER COMPLEXED WITH MJ33, SULFATE, AND CALCIUM
1L8S 2002-12-25 1.55 CARBOXYLIC ESTER HYDROLASE COMPLEX (DIMERIC PLA2 + LPC-ether + ACETATE + PHOSPHATE IONS)
1FXF 2001-09-25 1.85 CARBOXYLIC ESTER HYDROLASE COMPLEX (DIMERIC PLA2 + MJ33 INHIBITOR + PHOSPHATE IONS)
2B00 2006-11-14 1.85 Crystal Structure of Porcine Pancreatic Phospholipase A2 in Complex with Glycocholate
2AZY 2006-11-14 1.9 Crystal Structure of Porcine Pancreatic Phospholipase A2 in Complex with Cholate
1FX9 2001-09-25 2.0 CARBOXYLIC ESTER HYDROLASE COMPLEX (DIMERIC PLA2 + MJ33 INHIBITOR + SULPHATE IONS)
1Y6O 2005-03-22 2.0 Crystal structure of disulfide engineered porcine pancreatic phospholipase A2 to group-X isozyme in complex with inhibitor MJ33 and phosphate ions
3P2P 1990-01-15 2.1 ENHANCED ACTIVITY AND ALTERED SPECIFICITY OF PHOSPHOLIPASE A2 BY DELETION OF A SURFACE LOOP
2AZZ 2006-11-14 2.2 Crystal Structure of Porcine Pancreatic Phospholipase A2 in Complex with Taurocholate
2B01 2006-11-14 2.2 Crystal Structure of Porcine Pancreatic Phospholipase A2 in Complex with Taurochenodeoxycholate
2PHI 1993-07-15 2.2 A LARGE CONFORMATIONAL CHANGE IS FOUND IN THE CRYSTAL STRUCTURE OF THE PORCINE PANCREATIC PHOSPHOLIPASE A2 POINT MUTANT F63V
1Y6P 2005-03-22 2.25 Crystal structure of disulfide engineered porcine pancratic phospholipase a2 to group-x isozyme
2B03 2006-11-14 2.3 Crystal Structure of Porcine Pancreatic Phospholipase A2 in Complex with Taurochenodeoxycholate
4DBK 2012-01-25 2.3 Crystal structure of porcine pancreatic phospholipase A2 complexed with berberine
3FVJ 2010-03-16 2.3 Crystal structure of phospholipase A2 1B crystallized in the presence of octyl sulfate
4G5I 2012-11-07 2.4 Crystal Structure of Porcine pancreatic PlA2 in complex with DBP
4P2P 1992-01-15 2.4 AN INDEPENDENT CRYSTALLOGRAPHIC REFINEMENT OF PORCINE PHOSPHOLIPASE A2 AT 2.4 ANGSTROMS RESOLUTION
3L30 2010-01-26 2.4 Crystal structure of porcine pancreatic phospholipase A2 complexed with dihydroxyberberine
5P2P 1991-10-15 2.4 X-RAY STRUCTURE OF PHOSPHOLIPASE A2 COMPLEXED WITH A SUBSTRATE-DERIVED INHIBITOR
3HSW 2009-06-30 2.5 Crystal Structure of Porcine Pancreatic Phospholipase A2 in Complex with 2-methoxycyclohexa-2-5-diene-1,4-dione
2B04 2006-11-14 2.5 Crystal Structure of Porcine Pancreatic Phospholipase A2 in Complex with Glycochenodeoxycholate
4O1Y 2014-01-29 2.5 Crystal structure of Porcine Pancreatic Phospholipase A2 in complex with 1-Naphthaleneacetic acid
3QLM 2011-04-06 2.5 Crystal structure of porcine pancreatic phospholipase A2 in complex with n-hexadecanoic acid
3O4M 2010-08-25 2.5 Crystal structure of porcine pancreatic phospholipase A2 in complex with 1,2-dihydroxybenzene
1P2P 1983-09-15 2.6 STRUCTURE OF PORCINE PANCREATIC PHOSPHOLIPASE A2 AT 2.6 ANGSTROMS RESOLUTION AND COMPARISON WITH BOVINE PHOSPHOLIPASE A2
3FVI 2010-03-16 2.7 Crystal Structure of Complex of Phospholipase A2 with Octyl Sulfates

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