INVESTIGADORES
IGLESIAS Alberto Alvaro
congresos y reuniones científicas
Título:
Unmasking the High Affinity of Escherichia coli Glycogen Synthase Towards its Polyglucan Substrate
Autor/es:
A.A. IGLESIAS; M. ALEANZI; M.D. ASENCIÓN DIEZ; M. MACHTEY; A. YEP; M.A. BALLICORA
Lugar:
Chicago
Reunión:
Congreso; ASBMB Annual Meeting; 2017
Institución organizadora:
ASBMB
Resumen:
In bacteria, glycogen synthesis occurs by a 3-step pathway where the glucosyl donor ADP-glucose synthesized by ADP-glucose pyrophosphorylase (EC 2.7.7.27) is used to elongate an α-1,4-glucan chain by glycogen-synthase (EC 2.4.1.21; GlgSase), following by the action of polyglucan branching enzyme (EC 2.4.1.18). The pathway was extensively studied in Escherichia coli where the three enzymes were structurally and functionally characterized. The E. coli GlgSase is an enzyme with a relatively high specific activity (~500 U/mg) but an apparent low affinity towards glycogen, as judged by Km values in the order of 0.65 mg/ml. Interestingly, site-directed mutants of the E. coli GlgSase with highly reduced Vmax decreased the Km. To have a Km equal to Ks requires the assumption that the enzyme is under rapid equilibrium, where kcat is negligible compared to the dissociation of substrate koff. This scenario sustains the hypothesis that a real (high) affinity towards glycogen by E. coli GlgSase could be masked in the kinetic measurement of Km by a relatively high kcat. It is important to note that the accurate determination of the actual affinity for glycogen is relevant to understand the enzyme action mechanism. For that reason, we conducted saturation curves of glycogen at different temperatures in order to analyze changes in Km under conditions where kcat is significantly reduced. We found that a decrease in one order of magnitude in the Vmax correlated with a ~200-fold reduction in Km. This behavior was also observed for GlgSase mutants (H161A and K305A) and when the wild type enzyme used alternative (to ADP-Glc) substrates. In addition, the high affinity of the enzyme for glycogen was confirmed by measurements of binding in the absence of catalysis, where Ks was determined to be in the order of μg/ml. This suggests that the kinetics of this enzyme could be better described by steady state models rather than rapid equilibrium.