Characterization of a cold-adapted glutathione synthetase from the psychrophile Pseudoalteromonas haloplanktis

Abstract

Glutathione (GSH) biosynthesis occurs through two ATP-dependent reactions, usually involving distinct enzymes; in the second step of this process, catalysed by glutathione synthetase (GshB), GSH is formed from γ-glutamylcysteine and glycine. A recombinant form of GshB from the cold-adapted source Pseudoalteromonas haloplanktis (rPhGshB) was purified and characterised. The enzyme formed a disulfide adduct with β-mercaptoethanol, when purified in the presence of this reducing agent. The homotetrameric form of rPhGshB observed at high protein concentration disassembled into two homodimers at low concentration. A new method for directly determining the rPhGshB activity was developed, based on [γ-³²P]ATP hydrolysis coupled to the GSH synthesis. The ATPase activity required the presence of both γ-glutamylcysteine and glycine and its optimum was reached in the 7.4–8.6 pH range; a divalent cation was absolutely required for the activity, whereas monovalent cations were dispensable. rPhGshB was active at low temperatures and had a similar affinity for ATP (K_m 0.26 mM) and γ-glutamylcysteine (K_m 0.25 mM); a lower affinity was measured for glycine (K_m 0.75 mM). The oxidised form of glutathione (GSSG) acted as an irreversible inhibitor of rPhGshB (K_i 10.7 mM) and formed disulfide adducts with the enzyme. rPhGshB displayed a great temperature-dependent increase in its activity with an unusually high value of energy of activation (75 kJ mol⁻¹) for a psychrophilic enzyme. The enzyme was moderately thermostable, its half inactivation temperature being 50.5 °C after 10 min exposure. The energy of activation of the heat inactivation process was 208 kJ mol⁻¹. To our knowledge, this is the first contribution to the characterization of a GshB from cold-adapted sources.