Fluorescence stopped-flow studies on the binding of 1,N6-etheno-NAD to bovine liver glutamate dehydrogenase.
Fluorescence stopped-flow techniques have been used to investigate the binding of the oxidised coenzyme eNAD to bovine liver glutamate dehydrogenase (L-glutamate:NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3) saturated with glutarate, a substrate analogue, by following the transient kinetics of fluorescence intensity changes associated with changes in the binding of 1,N6-etheno-NAD (eNAD) to the enzyme, using displacement by NAD, NADP, ADP or GTP. Computer simulations of the various kinetic models provide a detailed picture of the molecular interactions between the active site (site I) and regulatory sites (sites II and III), specific for adenine and guanine nucleotides, respectively. The observed enhancement of the eNAD dissociation rate constant from site I can satisfactorily be accounted for as being due to the effect of ADP or NAD (and to a lesser extent NADP) binding to site II. This provides a mechanism for the allosteric activation of this enzyme via a predominantly intrasubunit interaction. By contrast the isomerisation of the enzyme induced by ADP alone is markedly slowed down by the occupancy of site I by eNAD in the presence of glutarate. The inhibitory effect of the allosteric effector GTP correlates with a tightening of eNAD binding, causing a decrease of the coenzyme dissociation rate constant followed by a slow isomerisation of the enzyme complexed with eNAD and glutarate.
Favilla
R
R
Department of Physics, University of Parma, Italy.
Martin
S R
SR
Bayley
P M
PM
D000244:Adenosine Diphosphate / Q000378:metabolism; D000465:Algorithms; D000818:Animals; D002417:Cattle; D005453:Fluorescence; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D005977:Glutarates / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000031:analogs & derivatives* / Q000378:metabolism
D005977:Glutarates; D009243:NAD; C011038:nicotinamide 1,N(6)-ethenoadenine dinucleotide; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D005969:Glutamate Dehydrogenase