Steady-state kinetics and transient studies of substrate and coenzyme analogue binding to clostridial glutamate dehydrogenase (GDH) during oxidative deamination

Abstract

to GDH and GDH-LGIu binary complex in 50mM phosphate buffer at pH 7.0 was determined by stopped-flow spectroscopy. The binding was monitored by measuring the fluorescence above 420nm, due to excitation at 294nm, using a cut-off filter. The binding of ENAD+ to GDH caused a monophasic enhancement in the fluorescence which was fitted to a single exponential function. The corresponding apparent first-order rate constants for different ENAD+ concentrations increased to a saturable level (Fig. la) and the data were fitted to a hyperbola indicating an apparent dissociation constant of 3 9 ( + 6 ) p ~ The binding of ENAD+ to GDH-LGlu binary complex also caused a monophasic enhancement in fluorescence but, in this case, followed by a linear phase, and thus the data were fitted to a single exponential followed by a steady-state process. The rate of the ensuing steady-state portion of the binding curve of ENAD+ to GDH-LGlu (0.124+0.004~-~) was comparable to the linear change in nucleotide fluorescence (excitation 340nm, emission 1420nm) due to the turnover of the substrates (0.153+0.001s-~). The binding of ENAD+ to the GDH-LGlu binary complex was also characterized by a hyperbolic increase in the rate of formation of the ternary complex with increasing coenzyme analogue concentration (Fig 1 b), yielding an apparent dissociation constant of 1 1 (52)pM. The rate of the enhancement in fluorescence of the binding of ENAD+ to either GDH or GDH-LGlu binary complex increased in an apparently hyperbolic manner and this behaviour indicates that there must be at least two steps involved in the formation of both binary and ternary complexes. These results point to a mechanism in which there is an isomerisation of the enzyme complex following the binding of ENAD'. The two conformational isomers of the GDH-ENAD+ binary complex or of the GDH-LGlu-&NAD+ ternary complex differ presumably in the binding mode of the coenzyme molecule. Since at 25OC in neutral aqueous solution 45(?5)% of ENAD+ is in the stacked form [3], it is possible that after initial binding the &-adenosine moiety in the extended conformation of ENAD' comes into close proximity with a tryptophan, probably W243 near the coenzyme binding site [4] in the protein, which would quench the fluorescence of the latter moiety of the coenzyme analogue The K, was assessed by measuring nucleotide fluorescence under steady-state conditions in which the concentration of LGlu was kept constant at 5 O m M (mixing chamber concentration) and concentration of ENAD+ varied. The dependence of the linear initial rates on ENAD+ concentration showed a hyperbolic increase followed by an inhibition at high concentrations (data not shown). Accordingly, steady-state parameters were determined by fitting the data to an equation for substrate inhibition giving a K, value of