Hemocyanin conformational changes associated with SDS-induced phenol oxidase activation

Abstract

The enzymatic activity of phenoloxidase is assayed routinely in the presence of SDS. Similar assay conditions elicit phenoloxidase activity in another type 3 copper protein, namely hemocyanin, which normally functions as an oxygen carrier. The nature of the conformational changes induced in type 3 copper proteins by the denaturant SDS is unknown. This comparative study demonstrates that arthropod hemocyanins can be converted from being an oxygen carrier to a form which exhibits phenoloxidase activity by incubation with SDS, with accompanying changes in secondary and tertiary structure. Structural characterisation, using various biophysical methods, suggests that the micellar form of SDS is required to induce optimal conformational transitions in the protein which may result in opening a channel to the di-copper centre allowing bulky phenolic substrates access to the catalytic site.

Hemocyanin from shrimp Litopenaeus vannamei shows hemolytic activity

“…Dodecamer is required for agglutination of Litopenaeus vannamei hemocyanin with bacterial cells and red blood cells [31]. Baird et al reported that the micellar form of SDS is required to induce optimal conformational transitions in hemocyanin, which may result in eliciting phenoloxidase activity [32]. Garcia-Carreno et al indicated that activation of hemocyanin to phenoloxidase by endogenous (serine proteinases) and exogenous (SDS) effectors needs complex aggregation for displaying enzyme activity [33].…”

Mechanistic studies of the tyrosinase-catalyzed oxidative cyclocondensation of 2-aminophenol to 2-aminophenoxazin-3-one

“…Therein, multiple metal ion binding modes for this structure were described through a cooperative model describing high copper ion plasticity controlling reactivity and substrate selectivity of the active site coppers is thought to limit CO to diphenolase activity and Hc to reversible oxygen binding. In support of copper ion plasticity and sampling between different enzyme forms, the reversible oxygen binding with Hc was altered through the addition of a denaturant (SDS) that moved blocking residues opening the active site to the catalytic oxidation of the diphenolic substrate dopamine while CO has copper coordinating histidine residues positioned in a conformationally labile loop regions [60, 61]. Based on our molecular modeling studies, the proposed binding orientations for 2-aminophenol and catechol are essentially superimposable with respect to benzene ring positioning near H296 and proximity to the CuA – CuB binding domains for both E met and E oxy forms of abTy (Figures 6 and 7).…”

Influence of limited proteolysis, detergent treatment and lyophilization on the phenoloxidase activity of Rapana thomasiana hemocyanin

“…Recently, a detailed study on arthropod Hc conformational changes associated with SDS-induced PO activation was reported [14]. It was shown that the micellar form of SDS (present at concentrations >1 mM in 100 mM sodium phosphate buffer, pH 7.5) is required to induce optimal conformational transitions in the protein, which may result in opening a channel to the di-copper centre allowing bulky phenolic substrates to access the catalytic site.…”

“…The kinetic parameters [K M (mM), V max (nmol min −1 mg −1 ) and k cat (min −1 ; expressed per copper pair)] were derived from non-linear regression data analysis of the dependence of the initial rates on the substrate concentration using HYPER software, taking into account the molar absorption coefficient of o-quinone 1417 M −1 cm −1 [11] or of dopachrome 3600 M −1 cm −1 [14] and the molecular mass of a FU (entity with one active site; 50 kDa).…”

“…The influence of limited proteolysis and of sodium dodecyl sulphate (SDS), known as an artificial activator of phenoloxidases and of phenoloxidase activity in arthropodan Hcs [14], is studied. Also the effect of lyophilization of the protein on the enzyme activity is investigated.…”