Steady-State kinetic study of substrate inhibition and product activation in the ADP-dependent phosphofructokinase/glucokinase from Methanococcus maripaludis

G. VALLEJOS; SB. KAUFMAN; RM. GONZÁLEZ LEBRERO; V. CASTRO-FERNANDEZ; V. GUIXÉ

Congreso; XL Reunión Anual de la Sociedad de Bioquímica y Biología Molecular; 2017

p { margin-bottom: 0cm; direction: ltr; color: rgb(0, 0, 0); text-align: justify; }p.western { font-family: "Times New Roman",serif; font-size: 12pt; }p.cjk { font-family: "Times New Roman",serif; font-size: 12pt; }p.ctl { font-family: "Times New Roman",serif; font-size: 10pt; }a:link { color: rgb(0, 0, 255); }p { margin-bottom: 0.25cm; direction: ltr; color: rgb(0, 0, 10); line-height: 120%; text-align: left; }p.western { font-family: "Calibri",serif; font-size: 11pt; }p.cjk { font-family: "Droid Sans Fallback"; font-size: 11pt; }p.ctl { font-family: "Times New Roman"; font-size: 11pt; }Insome archaeal glycolysis, glucokinase and phosphofructokinaseactivities utilize ADP instead of ATP as phosphoryl donor. Inorganisms belonging to Methanococcales one enzyme performs bothactivities. Until now, all these enzymes have been reported asnonregulated. Nevertheless, recently we found that the bifunctionalPFK/GK enzyme from Methanococcus maripaludis (MmPFK/GK) is activatedby AMP (reaction product) and also presents substrate inhibition byglucose.Inthis work, GK activity was measured at different [glucose] at severalfixed MgADP and AMP concentrations. Analysis of these experimentsshows a substrate inhibition effect when glucose increase above 100mM, and this inhibition effect is enhanced with an increment of[AMP].Inorder to obtain Vmax, KM and KI values for each fixed [MgADP] and[AMP], a canonical substrate inhibition model was used. Based on theanalysis of Vm and Km as a function of [MgADP] at fixed [AMP], anordered sequential mechanism was assigned, with MgADP as the firstsubstrate to bond. Additionally, substrate inhibition can beexplained by formation of a dead-end complex between free enzyme andglucose. Atfixed [MgADP], an increase of [AMP] causes a hyperbolic decrease ofboth Km and KI for glucose. Taking this into account, we concludethat activation by AMP of MmPFK/GK occurs mainly through amodification of glucose affinity. Also, the increment of substrateinhibition produced by AMP can be explained as an increase in glucoseaffinity of the free enzyme, driving the formation of thenonproductive enzyme-glucose complex.