Interfacial catalysis by phospholipase A2: dissociation constants for calcium, substrate, products, and competitive inhibitors

Abstract

Interpretation of the kinetics of interfacial catalysis in the scooting mode as developed in the first paper of this series [Berg et al. (1991) Biochemistry 30 (first paper of six in this issue)], was based on the binding equilibrium for a ligand to the catalytic site of phospholipase A2. In this paper, we describe direct methods to determine the value of the Michaelis-Menten constant (KMS) for the substrate, as well as the equilibrium dissociation constants for ligands (KL) such as inhibitors (KI), products (KP), calcium (KCa), and substrate analogues (KS) bound to the catalytic site of phospholipase A2 at the interface. The KL values were obtained by monitoring the susceptibility to alkylation of His-48 at the catalytic site of pig pancreatic PLA2 bound to micellar dispersions of the neutral diluent 2-hexadecyl-sn-glycero-3-phosphocholine. The binding of the enzyme to dispersions of this amphiphile alone had little effect on the inactivation rate. The half-time for inactivation of the enzyme bound to micelles of the neutral diluent depended not only on the nature of the alkylating agent but also on the structure and the mole fraction of other ligands at the interface. The KL values for ligands obtained from the protection studies were in excellent accord with those obtained by monitoring the activation or inhibition of hydrolysis of vesicles of 1,2-dimyristoyl-sn-glycerophosphomethanol. Since only calcium, competitive inhibitors, and substrate analogues protected phospholipase A2 from alkylation, this protocol offered an unequivocal method to discern active-site-directed inhibitors from nonspecific inhibitors of PLA2, such as local anesthetics, phenothiazines, mepacrine, peptides related to lipocortin, 7,7-dimethyleicosadienoic acid, quinacrine, and aristolochic acid, all of which did not have any effect on the kinetics of alkylation nor did they inhibit the catalysis in the scooting mode.

Altered lung phospholipid metabolism in mice with targeted deletion of lysosomal-type phospholipase A2

“…Based on this finding, we proposed that the degradation of DPPC in the lung epithelium was attributable in large part to the activity of a lysosomal type of PLA 2 . However, MJ33 is known to inhibit other PLA 2 activities (7,28), so the result of these inhibitor studies could not be considered definitive. In the present study, we have continued the evaluation of the effects of aiPLA 2 on lung DPPC metabolism using mice lacking Prdx6 generated by targeting of the Prdx6 gene (13).…”

“…MJ33, a transition-state analog competitive inhibitor of PLA 2 (7), inhibited the degradation of DPPC by ‫ف‬ 50% in both the whole lung and isolated alveolar type 2 cells (3,6). In vitro measurement of PLA 2 activity in homogenate of lungs or isolated type 2 cells was markedly inhibited by MJ33 when assayed at pH 4.0 in Ca 2 ϩ -free medium but had no effect on Ca 2 ϩ -dependent PLA 2 activity at pH 8.5 (3,6).…”

Phospholipase A2 Engineering. Structural and Functional Roles of the Highly Conserved Active Site Residue Aspartate-49

“…The equilibrium dissociation constants for active site-directed ligands (K L *, where L ) P, S, I, or Ca) bound to the active site of PLA2 at the interface were determined by monitoring the rate of alkylation of His-48 by p-nitrophenacyl bromide as described elsewhere (Jain et al, 1991a;Yu et al, 1993), except that phenacyl bromide was used with the triple mutant. For WT and most mutants including D99N the alkylation time for the enzyme in the aqueous phase was typically <5 min with p-nitrophenacyl bromide (2 mM); however, for the triple mutant the alkylation time was over 25 min.…”

“…The remaining PLA2 activity of each protection reaction in the presence of ligands was detected as a function of time, and the results are plotted in accordance with the ScruttonUtter equation to obtain values of the various equilibrium dissociation constants (Jain et al, 1991a;Yu et al, 1993. The uncertainty of these derived values is typically (20%; however, for the triple mutant uncertainty in the 2 Definition of kinetic parameters at the interface: KCa*, dissociation constant for Ca 2+ determined by the protection method; KCa*(S), effective dissociation constant for Ca 2+ under catalytic conditions at a mole fraction of 1 of the substrate; KI*, dissociation constant of inhibitor; KM*, Michaelis constant; KP*, dissociation constant of product; KS*, dissociation constant of substrate; kcat, turnover number at saturating substrate concentration; NSki, apparent second-order rate constant; Vo, turnover number at XS ) 1; XI, mole fraction of inhibitor.…”

A High-Throughput Screen for Endothelial Lipase Using HDL as Substrate

“…If a compound binds in the active site, the rate of alkylation is reduced, whereas compounds binding elsewhere have no effect. 26 In the case of EL, tetrahydrolipstatin (THL; Orlistat) was used for the protection experiments. THL is a time-dependent, irreversible inhibitor of several lipases 27 and acts by acylating the active site serine of these enzymes.…”