28 research outputs found
Large-Scale Production of Human Glioblastoma-Derived Cancer Stem Cell Tissue in Suspension Bioreactors to Facilitate the Development of Novel Oncolytic Therapeutics
Multiple ligand binding and kinetic isotope effects in methanol dehydrogenase
The reaction of PQQ-dependent methanol dehydrogenase (MDH) has been studies by steady-state and stopped-flow kinetic methods, with particular reference to multiple ligand binding and kinetic isotope effects (KIEs). Phenazine ethosulphate (PES; an artificial electron acceptor) and cyanide (a suppressant of endogenous activity), but not ammonium (an activator of MDH), compete for binding at the catalytic methanol-binding site. MDH activity is dependent on activation by ammonium, bit is inhibited at high ammonium concentrations. Glycine ethyl ester (GEE; an alternative activator) does not inhibit enzyme activity to the same degree as ammonium. Data suggest a close spatial relationship between the Ks and Ki activator-binding sites such that binding of GEE to the Ks site sterically impairs binding of GEE to the Ki site. Cyanide is less effective at suppressing endogenous activity with GEE, which is attributed to impaired binding of cyanide at the catalytic site through steric interaction with GEE at the Ks site. Combined, this data suggests that the catalytic methanol-binding site and the K s and Ki sites are in close proximity. PES and substrate influence the stimulatory and inhibitory effects of activator through competitive binding, giving rise to unusual KIEs as a function of activator and PES concentration. The KIEs are independent of temperature, consistent with the proposed competitive binding model. Stopped-flow studies indicate that at high ammonium concentrations, the oxidative half-reaction becomes more rate-limiting. With GEE, the oxidative half-reaction makes a larger contribution to rate limitation, stopped-flow method has enabled study of the reductive half-reaction of MDH. A kinetic mechanism for the reaction cycle of MDH is proposed. Furthermore, this study emphasises the need for caution in using KIEs, and the temperature dependence of KIEs, as a probe for enzymatic hydrogen tunnelling