The mechanism of substrate and coenzyme binding to clostridial glutamate dehydrogenase during oxidative deamination

Abstract

The binding of NAD' and L-Glutamate to glutamate dehydrogenase (GDH) from Clostridium symbiosum has been investigated by stopped-flow fluorescence spectroscopy. The formation of the binary complexes produces little change in the protein fluorescence but formation of the ternary complex results in quenching of its fluorescence with a maximum value of 40%. This finding, coupled with the finding that a step prior to hydride transfer but subsequent to ternary complex formation is rate limiting, has enabled us to monitor the kinetics of ternary complex formation in detail. The ternary complex can be formed via the GDH-NAD' or the GDH-L-GIu binary complexes, but the route via the GDH-NAD' binary complex is the preferred pathway. The equilibrium and rate constants for the formation of the two binary complexes and the ternary complex formed via the two possible pathways have been determined. These studies have revealed an interaction between the coenzyme-binding site and the substrate-binding site, which lead to a decrease in the binding constant for the second substrate binding to the enzyme. The free energy coupling between the binary and ternary complexes is about 2.4-2.8 kJ mol-'. We propose that there is a further isomerisation of the ternary complex, which is rate limiting for the steady-state turnover of the enzyme. Formation of this complex is characterised by an increased negative interaction, with a free energy coupling between these complexes of 6.3-11.6 kJ . mol-'.Glutamate dehydrogenase (GDH) catalyses the reversible amination of 2-oxoglutarate by ammonia to L-Glutamate, using nicotinamide coenzymes as follows:This reaction is central to the metabolism of both higher organisms, which need to dispose of excess nitrogen, and many lower organisms, which are able to assimilate nitrogen in the form of ammonia. Accordingly, GDH enzymes from a variety of sources have been isolated and studied (Goldin and Frieden, 1971 ;Smith et al., 1975). Mechanistically, the GDH studied in most detail is that from beef liver mitochondria. This enzyme has been studied using steady-state kinetic, transient kinetic and isotope equilibrium exchange methods in an attempt to elucidate the mechanism (e.g. Frieden, 1959a and b; Dalziel, 1969, 1970;Silverstein and Sulebele, 1973;Pantaloni and Iwatsubo, 1967;di Franco and Iwatsubo, 1972;Chen and Engel, 1975; Rife and Cleland, 1980;Fisher et al., 1987). However, an overall view of its mechanism is still elusive. With regard to the order of substrate binding, the only generally accepted fact is that GDH follow a sequential mechanism, in which a central complex exists with all the substrates simultaneously present. Analysis of the steady-state kinetics for oxidative deamination is often complicated by non-classical behaviour, usually attributed to allosteric interactions (Frieden, 1959a;Monod et al., 1963 ;Engel and Dalziel, 1969;Goldin and Frieden, 1971). In the case of beef GDH, saturation with the allosteric effector ADP simplifies the kinetic behaviour, The steady-state analysis also s...

An enzymatic process to α-ketoglutarate from l-glutamate: the coupled system l-glutamate dehydrogenase/NADH oxidase

“…In accordance with the literature, 7,8,15,16 we found L L-gluDH to be inhibited by all three products of the oxidative deamination reaction, ammonium ion, 15,16 a-ketoglutarate, and NADH.…”

Crystallographic and Pre-steady-state Kinetics Studies on Binding of NADH to Wild-type and Isoniazid-resistant Enoyl-ACP(CoA) Reductase Enzymes from Mycobacterium tuberculosis

“…The emitted light was selected with a WG420 Scott filter, positioned between the photomultiplier and the sample cell as described. 57,58 Data were stored on an Acorn A5000 computer, and analyzed by non-linear regression. Data acquisition was carried out using a split time base (20 and 200 ms) with the first half of data acquired over 10% of the time period monitored for the second half of the split time base.…”

On the mechanism of human stefin B folding: II. Folding from GuHCl unfolded, TFE denatured, acid denatured, and acid intermediate states

“…7 Additional changes in the far UV CD signal may accompany changes in the tertiary structure, which usually occur much later, and are seen in the near UV CD. Fluorescence measurements were found especially useful as they demand relatively low quantities of the protein, and the modern stopped-flow fluorimeters have dead times below 1.2 ms, 8 which allows studies of some of the earliest phases in folding. In addition to the intrinsic protein fluorescence, the changes in fluorescence of the hydrophobic probe, 1-anilino-naphthalene-8-sulfonate (ANS), have been used [9][10][11] for folding studies.…”