The essentail active‐site lysines of clostridial glutamate dehydrogenase

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Abstract
Glutamate dehydrogenase (GDH) of Clostridium symbiosum, like GDH from other species, is inactivated by pyridoxal 5′‐phosphate (pyridoxal‐P). This inactivation follows a similar pattern to that for beed liver GDH, in which a non‐covalent GDH‐pyridoxal‐P complex reacts slowly to form a covalent complex in which pyridoxal‐P is in a Schiff's‐base linkage to lysine residues. image The equilibrium constant of this first‐order reaction on the enzyme surface determines the final extent of inactivation observed [S. S. Chen and P. C. Engel (1975) Biochem. J. 147, 351–358]. For clostridial GDH, the maximal inactivaton obtained was about 70%, reached after 10 min with 7mM pyridoxal‐P pH 7. In keeping with the model, inactivation became irreversible after reduction with NaBH4. The NaBH4‐reudce enzyme showed a new absorption peak at 325 nm. K m values for NAD+ and glutamate were unalterd, although Vmax values were decreased by 70%. Kinectic analysis of the inactivation gave values of 0.81 ± 0.34 min−1 for K3 and 3.61 ± 0.95 mM for K2/K1. Thelineat plot of 1/(1‐R) against 1/[pyridoxal‐P], where R is the limiting residual activity reached in an inactivation reaction, gave a slightly higher valus for K2/K1 of 4.8 ± 0.47 mM and K4 of 0.16 ± 0.01 min−1.NADH, NAD+, 2‐oxoglutrate, glutarate and succinate gave partial protection against inactivation, the biggest effect being that of 40 mM succinate (68% activity compared with 33% in the control). Paired combinations of glutarate or 2‐oxoglutarate and NAD+ gave slightly better protection than the separate components, but the most effective combination was 40mM 2‐oxoglutarate with 1 mM NADH (85% activity at equilibrium). 70% inactivated enzyme showed an incorporation of 0.7 mM pyridoxal‐P/mol subunit, estimated spectrophotometrically after NaBH4 reduction, in keeping with the 1:1 stoichiometry for the inactivation. In a sample protected with 2‐oxoglutarate and NADH, however, incorporation was 0.45mol/mol, as against 0.15mol/mol expected (85% active).Tryptic peptides of the enzyme, modified with and without protection, were purified by HPLC. Two major peaks containing phosphopyridoxyllysine were unique to the unprotected enzyme. These peaks yielded three peptide sequences clearly homologous to sequences of other GDH species. In each case, a gap at which no obvious phenythiohydantion‐amino‐acid was detected, matched a tryptic cleavage. Modification of these three lysines, corresponding to Lys92, Lys116 and Lys128 in Escherichia coli GSH, appears to be mutually exclusive and modification of any one of them abolishes activity. Comparison with the cystal structure indicated that these lysines are clustered in the activesite cleft and likely to interact with the dicarboxylate substrate.