Study of the effect of lanthanide ions on the kinetics of glutamate dehydrogenase by a chronoamperometric method

Abstract

A chronoamperometric method, based on the oxidation of reduced nicotinamide adenine dinucleotide (NADH) on a glassy carbon electrode, for assaying glutamate dehydrogenase activity is described. Once the reaction was initiated by addition of enzyme into a well stirred assay mixture containing NADH, the concentration of NADH and the steady-state limiting current of NADH oxidation on the electrode decreased linearly in a short time range from the start of the reaction. The rate of the enzyme-catalysed reaction can be calculated from the decrease in the current. This method is reliable for the accurate determination of enzyme activity in purified extracts from liver and crude extracts from cucumber root. The major advantage of this method is the real-time and continuous measurement of the change in coenzyme concentration, so the real initial reaction rate, VO, can be determined. This method has been used to detect the effect of La3+, Eu3+, Yb3+ and Zn2+ ions on the enzyme activity. The results suggest that lanthanide ions can increase the activity of the enzyme in glutamate synthesis, and Zn2+ acts as a potent inhibitor of the enzyme.

Structural features of aluminium(III) complexes with bioligands in glutamate dehydrogenase reaction system – A review

“…With the increase of Al(III) concentrations (30- 80 lM), the enzyme GDH activity was activated. It was known that the step of dissociation of the NAD + ÀGDH complex is the rate-limiting step in the reductive amination reaction [35]. However, our voltammetry studies indicated that Al(III) could interrupt the binding of NAD + to GDH when the concentration of metal ion is increased.…”

“…Electrochemical analysis has been recognized as a powerful tool for measuring trace metal ions and biomolecules in the biological system [35]. Its remarkable sensitivity, simple instrumentation, low cost, simple operation procedure, fast and reliable results and many other advantages make it satisfactory for many biological system analyses [36,37].…”

Inhibition by N′-nitrosonornicotine of the catalytic activity of glutamate dehydrogenase in α-ketoglutarate amination

“…One of the few convective electrode systems is the rotating disk electrode. Based on the diffusion layer approach described by Bard and Faulkenr,[19] Xin and Gao [16] developed a chronoamperometric method using a disk electrode in a well-stirred solution. It is assumed that convection maintains a uniform concentration of all species that are equal to the bulk values up to a certain distance, i.e., the "diffusion" layer thickness (d) at the electrode fed by convective transfer.…”

“…In addition, NADH may be monitored by oxidation at an electrode surface and this oxidation reaction has well been studied [13 -15]. In 1996, Xin and Gao proposed a chronoamperometric method to evaluate the initial kinetics of the enzyme-catalyzed reaction [16] and their method represents a significant advance. One of the advantages of the method is that it could be used to obtain the real initial reaction rate.…”

Electrochemical Studies of the Inhibition and Activation Effects of Al (III) on the Activity of Bovine Liver Glutamate Dehydrogenase

“…Electrochemical analysis has always been recognized as a powerful tool for measuring trace metal ions and biomolecules in the biological system. Its remarkable sensitivity, simple instrumentation, lower costs, simple operation procedure, faster and reliable results and many other advantages make it satisfy many requires and extensively applied in different biological system analysis or in other fields [16]. In the past, we have investigated the interactions of Al (III) with the substrates (α-KG/ L-Glu) [17,18] and coenzyme I (NAD + /NADH) [19,20] of the GDH enzyme system.…”