Functional significance of the conformational dynamics of the N-terminal segment of secreted phospholipase A2 at the interface.
The kinetic and fluorescence properties of several pig pancreatic phospholipase A2 (PLA2) with substitutions and deletions in the N-terminal region and of tyrosines 52 and 73 are characterized. The substitutions Ala-1-D-Ala or -Gly, Trp-3-Phe, Gln-4-Nle, Arg-6-Glu, Tyr-52-Phe, and Tyr-73-Phe had at the most only a modest effect on the interfacial catalytic activity on the anionic interface to which they bind with high affinity. The observed rate of hydrolysis in the scooting mode by deletion mutants lacking one or more successive residues from the N-terminal region was lower by 50-95%. Detailed kinetic analysis of the deletion mutant lacking Ala-1 (des-1-AMPA) showed that the 50% decrease in the rate is due to a 5-fold increase in the interfacial Michaelis-Menten parameter, KM*, without a significant change in kcat. These results and direct measurements show that the primary effect of Ala-1 deletion is to lower the affinity for the active site directed ligands. Although the affinity of these mutants for anionic interface remains the same as for the wild type, the affinity for zwitterionic neutral diluents is considerably lower. Significant differences in the fluorescence quantum yields and the heterogeneity in the frequency-domain fluorescence intensity decays of these enzymes suggest that both in solution and at the interface the N-terminal region is an ensemble of conformations rather than a discrete state. Additional results suggest that the interfacial microenvironment of Trp-3 in des-1-AMPA is more polar and Trp-3 is more accessible to quenching by acrylamide.(ABSTRACT TRUNCATED AT 250 WORDS)
Maliwal
B P
BP
Department of Biological Chemistry, University of Maryland Medical School, Baltimore 21201.
Yu
B Z
BZ
Szmacinski
H
H
Squier
T
T
van Binsbergen
J
J
Slotboom
A J
AJ
Jain
M K
MK
D000818:Animals; D002384:Catalysis; D005454:Fluorescence Polarization; D017353:Gene Deletion; D006868:Hydrolysis; D007700:Kinetics; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry*; D010741:Phospholipases A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054467:Phospholipases A2; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013552:Swine
D010446:Peptide Fragments; D010741:Phospholipases A; D054467:Phospholipases A2