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
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.