Metal‐ion‐ and pH‐induced conformational changes of acutolysin D from Agkistrodon acutus venom probed by fluorescent spectroscopy

Abstract

Acutolysin D isolated from the venom of Agkistrodon acutus is a protein of 44 kDa with marked hemorrhagic and proteolytic activities. The metal-ion- and pH-induced conformational changes of acutolysin D have been studied by following fluorescence and activity measurements. Here we provide evidence for the fact that native holo-acutolysin D adopts two different conformations, native state a, stable in the weak acidic pH range from 5.5 to 7.0 with low activity, and native state b, stable in the weak alkaline pH range from 8.0 to 9.0 with high activity. Holo-acutolysin D has an optimum pH of 9.0 for caseinolytic activity and a maximum fluorescence at pH 9.0. The protein adopts the most stable conformation at pH 9.0. The addition of 1 mM Zn(2+) shifts both the alkali-induced unfolding transition curve and the alkali-induced inactivation curve toward higher pH value but has little effect on the acid-induced unfolding transition curve. No obvious effects on the pH-induced unfolding transition curve and the pH-dependent activity curve have been observed after the addition of 1 mM Ca(2+) to holo-acutolysin D. The results indicate that Zn(2+) is essential for its CA, while Ca(2+) is not essential for its CA. Removal of Ca(2+) and Zn(2+) from the protein enhances its sensitivity to pH and significantly reduces its overall stability during acid-induced denaturation. The kinetic results of the demetalization of holo-acutolysin D show that the demetalization rate constant K(1) for a slower reaction linearly decreases with the pH increase from 5.0 to 9.0, while K(2) for the faster reaction linearly increases with the pH change from 5.0 to 7.0. It is also evident from the present work that the free Zn(2+)-induced inactivation in the pH range from 8.0 to 9.0 should be attributed to the effect of Zn(OH)(2) precipitation on the protein.

Small Angle X-ray Scattering, Molecular Modeling, and Chemometric Studies from a Thrombin-Like (Lmr-47) Enzyme of Lachesis m. rhombeata Venom

“…Thus, according to the enzyme activity profile for different pH levels, it shows the highest activity at pH 8.0 [ 10 ]. Therefore, this finding follows previous observation in another system associated with its anti-coagulant function [ 23 , 24 ]. Similarly, using fluorescence spectroscopy, it was also not feasible to identify an intermediate state, possibly because the tryptophan and tyrosine residues are located in regions unaffected by pH variation.…”

“…Lmr-47 was purified as described before [ 23 ]. Briefly, the venom (100 mg) was equilibrat-281 ed in 50 mM Tris–HCl containing 0.5 M NaCl, pH 7.0, and subjected to affinity chromatography on a benzamidine–agarose column.…”

High-Resolution Crystal Structure of the Snake Venom Metalloproteinase BaP1 Complexed with a Peptidomimetic: Insight into Inhibitor Binding

“…Another possible mechanism for pH-dependent modulation of proteolytic activity is a more widespread change in enzyme structure (52). For the BaP1 3 inhibitor complex, there are only a few side chain and loop shifts, mainly at the protein surface and the active site, but no significant conformational changes in the peptide backbone at different pH values can be observed.…”

Caracterização dos efeitos locais e sistémicos da metaloprotease BtaHF purificada a partir do veneno total de Bothriopsis taeniata

“…A fração BtaHF mostra comportamento similar com outras SVMP de baixa massa molecular perante diversos valores de pH (Figura 13; Gutierrez et al, 1995b;Stroka et al, 2005;Bello et al, 2006;Torres-Huaco et al, 2010) (Wu et al, 2009). Em condições alcalinas a perda de atividade caseinolítica seria devido à perda do átomo de Zinco do sítio catalítico na forma de Zn(OH) 2 (Xu et al, 2004), este último processo ocorreria mais devagarosamente em comparação com o que ocorre em condições ácidas.…”

“…Íons divalentes são componentes importantes dos venenos ofídicos, e agem como cofatores de proteínas como as metaloproteases de veneno de serpente. Análises estruturais mostraram que as SVMP possuem Zn +2 , Ca +2 e Mg +2 como cofatores em proporção de 1:1, 2:1 e 1:1 g/mol de proteína, respectivamente, assim, o íon Zn +2 é essencial para a atividade enzimática das SVMP (Fox & Serrano, 2009) aumenta o ordenamento estrutural da proteína ao se ligar à região que se encontra oposta ao sítio catalítico (Gomis-Ruth et al, 1994), o qual produziria o aumento da atividade catalítica (Xu et al, 2004;Zhang et al, 2004). Contrariamente, os íons de Zn +2 e Mn +2 , mostraram efeito inibitório sobre a atividade caseinolítica da BtaHF (Figura 16A).…”