Spectroscopic investigation of phenolic groups ionization in the vipoxin neurotoxic phospholipase A(2): comparison with the X-ray structure in the region of the tyrosyl residues

The neurotoxin vipoxin is the major lethal component of the venom of Vipera ammodites meridionalis, the most toxic snake in Europe. It is a complex be tween a toxic phospholipase A(2) (PLA(2)) and a non-toxic protein inhibitor (Inh). Tyrosyl residues are involved in the catalytic site (Tyr 52 and 73) and in the substrate binding (Tyr 22). Spectroscopic studies demonstrated differences in the ionization behavior of the various phenolic hydroxyl gro ups in the toxic PLA(2). The tyrosyl side chains of the enzyme can be class ified into three groups: (a) three phenolic hydroxyls are accessible to the solvent and titrate normally, with a pK(eff) = 10.45; (b) three residues a re partially 'buried' and participate in hydrogen bonds with neighboring fu nctional groups. They titrate anomalously with a pK(eff) = 12.17; (c) two t yrosines with a pK(eff) = 13.23 are deeply 'buried' in the hydrophobic inte rior of PLA(2). They became accessible to the titrating agent only after al kaline denaturation of the protein molecule. The spectroscopic data are rel ated to the X-ray structure of the vipoxin PLA(2). The refined model was in vestigated in the region of the tyrosyl side chains. The accessible surface area of each tyrosyl residue and each phenolic hydroxyl group was calculat ed. A good correlation between the spectrophotometric and the crystallograp hic data was observed. The ionization behavior of the phenolic groups is ex plained by peculiarities of the protein three-dimensional structure and the participation of tyrosines in the catalytic site hydrogen bond network. At tempts are made to assign the calculated pK(eff) values to individual resid ues. The high degree of 'exposure' on the protein surface of Tyr 22 and 75 is probably important for their function as parts of the substrate binding and pharmacological sites. (C) 1999 Elsevier Science B.V. All rights reserv ed.