Phospholipase A2 isolated from Naja melanoleuca snake venom was modified with 4-chloro-3,5-dinitrobenzoate. The modification reaction was accelerated by the cofactor Ca2+. Micellar concentrations of the substrate analog n-tetradecylphosphocholine retarded the inactivation of the enzyme. Modification resulted in the incorporation of one mole dinitrobenzoate/mole of protein. The modified residue could be identified as Lys-6 in the amino acid sequence of the enzyme. Although the modified enzyme still retains a high affinity for micellar substrate analogs and the cofactor Ca2+, it had lost nearly all its activity towards micellar substrates. Upon reduction of the nitro groups in the modified protein the enzymatic activity could be restored by more than 50%. Therefore Lys-6 cannot be directly involved in enzymatic catalysis. It is concluded that introduction of the negatively charged dinitrobenzoate group on Lys-6 gives rise to a distortion of the active site of the enzyme.

The fluorescent probe 8-anilinonaphtalene-1 -sulfonate (ANS) binds at the active site of the Nuja rnelanoleuca snake venom phospholipase A,, thus protecting the enzyme against active-site-directed chemical modification. Both hydrophobic and electrostatic interactions are involved in the binding. At pH 7.5, a binding constant of 100pM was determined, which improved twofold upon addition of the enzymatic cofactor Ca2 +. The pH dependence of the ANS binding in the absence and presence of Ca2+ ions showed a perturbation of a group with a pK, value of 5.2, which could be assigned to the carboxylate group of the Ca2+-binding ligand Asp49 at the active site of the protein.Monomeric concentrations of the substrate analog n-decylphosphocholine displace ANS from the protein, indicating again that both ligands bind at the active site. Binding studies with several modified N . rnelanoleuca enzymes showed that a loss of enzymatic activity on aggregated substrates was correlated with a loss of affinity for the active site bound ANS molecule. It is suggested therefore, that the fluorescent ANS probe can detect structural rearrangements at the active site, which are important for enzymatic activity.8-Anilinonaphtalene-1-sulfonate (ANS) has been used extensively as a fluorescent probe to monitor conformational changes in proteins. Whereas in aqueous solution the probe shows only a weak green fluorescence, an intense blue-shifted fluorescence is observed upon interaction with several biological macromolecules or membranes. A decreased polarity of the probe's environment is thought to be the main factor contributing to the observed fluorescence enhancement upon binding of ANS to a protein [l].Phospholipase A, catalyzes the hydrolysis of the 2-acyl ester bonds in diacylphosphoglycerides. The enzyme needs Ca2+ as an absolute cofactor, which is bound at the active site of the protein. Pieterson et al. [2] showed that the fluorescence of ANS is increased and blue-shifted upon addition of the pig pancreatic phospholipase A,, indicating that this enzyme binds one or more ANS molecules. Since the addition of the cofactor Ca2+ to the ANS-protein complex results in a further increase of the ANS fluorescence, the probe could be used to determine a Ca2+-binding constant for this enzyme. Enzyme. Phospholipase A, (EC 3.1.1.4).Recently [6, 71, we have reported that certain chemical modifications in the N-terminal region of the polypeptide chain inactivate the Naja rnelanoleuca phospholipase A,. As the affinity for lipid-water interfaces of the modified proteins is hardly affected, the loss of enzymatic activity might be attributed to changes in the active site of the protein. It will be demonstrated in the present study that ANS binds at the active site, where the cofactor Ca2 + and monomeric substrates are bound. Moreover, it will be shown that ANS can be used as a fluorescent probe to detect conformational changes at the active site of the modified proteins. MATERIALS AND METHODS EnzymesPhospholipase A, was purified from Naja rnelunoleuca sna...

In phospholipase A, from Nuju melanoleuca snake venom all four lysines were converted into the 8-amidinated derivatives without reaction of the a-amino group. The amidinated phospholipase (AMPA) showed high enzymatic activity.Starting from AMPA, chemical modification reactions were carried out at the %-amino function. This group was blocked with a tert-butyloxycarbonyl or a phenylthiocarbamyl group. Furthermore the polypeptide chain was shortened by one residue by removing the N-terminal asparagine, resulting in the formation of des-Am1-AMPA. The native enzyme was shortened by eight residues by cyanogen bromide cleavage at the single methionine' residue.Although all modified proteins show a reduced affinity for monomeric lipids, they are easily saturated with micellar substrate analogs. Whereas the removal of the N-terminal octapeptide abolished all enzymatic activity the other modified enzymes possess a low (1 X), but measurable enzymatic activity. It is concluded that chemical modifications in the N-terminal region give rise to a distortion of the active site, thus reducing the activity of the lipid-bound enzyme.The enzyme phospholipase A, is ubiquitous in Nature. It can be isolated in large amounts from snake venoms and mammalian pancreas as a small (molecular mass, m, about 14 kDa) heat-stable enzyme. The enzymes from these sources catalyze the hydrolysis of the fatty acid ester bonds at the 2-position of diacyl-sn-3-phosphoglycerides for which Ca, + ions are required as an absolute cofactor. The physicochemical nature of the substrate has an enormous effect on the activity of phospholipase A,. Whereas a low enzymatic activity is observed on monomeric substrates, the activity is increased by several orders of magnitude upon passing the critical micelle concentration, when the enzyme forms a complex with the aggregated lipid substrate. Although the structural homology between snake venom and pancreatic phospholipases suggests a common catalytic mechanism, different models have been proposed to explain this interfacial activation (for a review, see Slotboom et al. [I]).Several studies have revealcd the importance of the N-terminal region of phospholipase A,. The affinity of the porcine pancreatic phospholipase A, for micellar substrate analogs is critically dependent upon a free positively charged N-terminal a-NH: group, which is locked in a buried position by an internal salt bridge. Even minor modifications result in a complete loss of enzymatic activity. The loss of enzymatic activity was attributed to a distortion of the lipid-binding domain of the modified enzyme [ 2 ] . Snake venom phospholipases also lose their enzymatic activity on micellar substrates after modification of the N-terminal amino acid residue [3,4], but, contrary to the modifiedpancreatic enzymes, the modified Abbreviations. DE 111, native phospholipase A, ; AMPA, c-amidinated phospholipase

Unlike porcine pancreatic phospholipase A,, the enzyme from Naja melanoleuca does not display biphasic kinetic behaviour at substrate concentrations around the critical inicelle concentration. This snake venom enzyme was further investigated by direct binding studies using n-tridecylphosphocholine. Binding of this substrate analog to the enzyme was monitored by using equilibrium gel filtration, equilibrium dialysis and ultraviolet difference spectroscopy. It is concluded that, in the presence of submicellar concentrations of n-tridecylphosphocholine, a lipid-protein complex is formed consisting of about 4 protein and 36 lipid molecules. CaZ + ions are required for the formation of this complex. A model is proposed which describes the formation of this type of complex. These lipidprotein aggregates are held responsible for the non-hyperbolic kinetic behaviour of the snake venom enzyme towards monomeric substrates.Phospholipase A, is widespread in Nature. The enzyme is abundant in pancreatic tissues as well as in the venoms of arthropods and snakes. Irrespective of the source, the enzymes show a high degree of homology. An identical mechanism for the hydrolysis of monomeric phospholipids has been postulated [I]. The activity of all phospholipases strongly depends on the physico-chemical state of the substrate. The enzyme displays Michaelis-Menten kinetics when acting on monomeric substrates. In the presence of certain organized lipidwater interfaces, phospholipase A, shows activities which can be three orders of magnitude higher than found for monomeric substrates. This results in a break in the velocity vs substrate concentration plots at the critical micelle concentration of the substrate [2].For the pancreatic enzymes it has been proposed by Verger et al. 131 that a particular surface region of the enzyme, called the interface recognition site (IRS), is responsible for the interaction with aggregated lipid-water interfaces.