FUNCTIONAL-SIGNIFICANCE OF THE CONFORMATIONAL DYNAMICS OF THE N-TERMINAL SEGMENT OF SECRETED PHOSPHOLIPASE-A2 AT THE INTERFACE

Citation
Bp. Maliwal et al., FUNCTIONAL-SIGNIFICANCE OF THE CONFORMATIONAL DYNAMICS OF THE N-TERMINAL SEGMENT OF SECRETED PHOSPHOLIPASE-A2 AT THE INTERFACE, Biochemistry, 33(15), 1994, pp. 4509-4516
Citations number
52
Categorie Soggetti
Biology
Journal title
ISSN journal
00062960
Volume
33
Issue
15
Year of publication
1994
Pages
4509 - 4516
Database
ISI
SICI code
0006-2960(1994)33:15<4509:FOTCDO>2.0.ZU;2-5
Abstract
The kinetic and fluorescence properties of several pig pancreatic phos pholipase A(2) (PLA2) with substitutions and deletions in the N-termin al region and of tyrosines 52 and 73 are characterized. The substituti ons 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 h igh affinity. The observed rate of hydrolysis in the scooting mode by deletion mutants lacking one or more successive residues from the N-te rminal region was lower by 50-95%. Detailed kinetic analysis of the de letion 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-M enten parameter, K-M, without a significant change in k(cat). These r esults and direct measurements show that the primary effect of Ala-1 d eletion 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 d iluents is considerably lower. Significant differences in the fluoresc ence quantum yields and the heterogeneity in the frequency-domain fluo rescence intensity decays of these enzymes suggest that both in soluti on and at the interface the N-terminal region is an ensemble of confor mations 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. This indicate s incomplete desolvation of the microinterface in case of des-1-AMPA. The frequency-domain anisotropy decays of Trp-3 indicate that the N-te rminal region of free PLA2 is somewhat rigid and becomes flexible as A la-1 and Leu-2 are deleted. On the other hand, on DTPM vesicles, the N -terminal region is essentially rigid in PLA2 and des-1-AMPA, and it i s only modestly flexible in bound des-1,2-AMPA. These observations dem onstrate a ligand-induced rigidification of the N-terminal region of t he EI form of PLA2, and it is surprising that such a rigidification i s observed in the absence of Ala-1. Taken together these results show that Ala-1 and the associated hydrogen-bond network affect the binding of an active site directed ligand via its role in desolvation of the interface but without a significant effect on the chemical step of the catalytic turnover cycle.