Química medicinal

Biochemical and Pharmacokinetic Properties of Aspergillus flavipes Glutathione-Homocystine Transhydrogenase of Unique Affinity for Homocystine Reduction using GSH as Hydrogen Donor

Abstract

Ashraf SA El-Sayed, Abdallaa E Hassan, Marwa A Yassin, Hend MM Ibrahim and Asmaa M Hassan

Glutathione-Homocystine Transhydrogenase (GHTHase) was characterized from Aspergillus flavipes as a novel enzymeof high specificity to reduce homocystine to homocysteine using GSH as hydrogen donor. GHTHase was further conjugated to mono-functional carboxyl polyethylene glycol (PEG) to improve its catalytic properties for various therapeutic uses especially against homocystinuria. The biochemical properties of free and PEG-GHTHase were assessed. The enzyme molecular mass was increased by 1.2 % (from 80 to 95 kDa) by PEG conjugation. The free and PEG-GHTHase have the same pH stability (6.5-8.0) and thermal stability (T1/2 1.0-1.3 h, at 50°C). Kinetically, the affinity and catalytic efficiency of PEG-GHTHase was decreased by 15% to GSH as hydrogen donor for reduction of homocystine than free enzyme. PEG-GHTHase has a slightly stability for suicide inhibitor as propargylglycine, hydroxylamine and iodoacetate. Upon proteolysis, the free enzyme retains less than 10% of its initial activity, comparing to 85.8 % of PEG-GHTHase upon trypsinand acid protease digestion for 30 min in vitro. From the pharmacokinetics properties in New Zealand rabbits, the half-life time of free and PEG-GHTHase was 10.0-12.2 h. Upon external infusion of NADH (20 μM) after 24 h of initial enzymes dosing, the half-life times of the enzymes were increased to 10 h. The biochemical and hematological parameters upon enzymes infusion were relatively not affected along the tested period. In vivo, GHTHase exhibits higher affinity to methionine and cysteine. Based on in vitro and in vivo biochemical properties, PEG-GHTHase could be a reliable enzyme in reduction of homocystine using GSH as electron donor. We assume, with more crystallographic studies, that GHTHase will be a new finding against various disorders-dependent homocystinuria.