ALTERATION OF THE AMINO-ACID SUBSTRATE-SPECIFICITY OF CLOSTRIDIAL GLUTAMATE-DEHYDROGENASE BY SITE-DIRECTED MUTAGENESIS OF AN ACTIVE-SITE LYSINE RESIDUE

Two residues, K89 and S380, thought to interact with the gamma-carboxy l group of the substrate L-glutamate, have been altered by site-direct ed mutagenesis of clostridial glutamate dehydrogenase (GDH). The singl e mutants K89L and S380V and the combined double mutant K89L/S380V wer e constructed. All three mutants were satisfactorily: overproduced in soluble form. However, only the K89L mutant was retained by the dye co lumn normally used in purifying the wild-type enzyme. All three mutant enzymes were purified to homogeneity and tested for substrate specifi city with 24 amino acids. The single mutant S380V showed no detectable activity. The alternative single mutant K89L, showed an activity towa rds L-glutamate that was decreased nearly 2000-fold compared with wild -type enzyme, whereas the activities towards the monocarboxylic substr ates alpha-aminobutyrate and norvaline were increased 2- to 3-fold. A similar level of activity was obtained with methionine (0.005 U/mg) an d norleucine (0.012 U/mg), neither of which give any activity with the wild-type enzyme under the same conditions. The double mutant showed decreased activity with all substrates compared with the wild-type GDH . In view of its novel activities, the K89L mutant was investigated in greater detail. A strictly linear relationship between reaction veloc ity and substrate concentration was observed up to 80 mM L-methionine and 200 mM L-norleucine, implying very high K-m values. Values of k(ca l)K(m) for L-methionine and L-norleucine were 6.7x10(-2) and 0.15 s(-1 )M(-1), respectively. Measurements with dithiobisnitrobenzoic acid sho wed that the mutant enzymes all reacted with a stoichiometry of one -S H group per subunit and all showed protection by coenzyme, indicating essentially unimpaired coenzyme binding. With glutamate or 2-oxoglutar ate as substrate the K-m values for the vestigial activity in the muta nt enzyme preparations were strikingly close to the wild-type K-m valu es. Both for wild-type GDH and K89L, L-glutamate gave competitive prod uct inhibition of 2-oxoglutarate reduction but did not inhibit the red uction of 2-oxocaproate catalysed by K89L enzyme. This suggests that t he low levels of glutamate/2-oxoglutarate activity shown by the mutant enzyme are due to trace contamination. Since stringent precautions we re taken, it appears possible that this reflects the level of reading error during overexpression of the mutant proteins. CD measurements in dicate that the S380V mutant has an altered conformation, whereas the K89L, enzyme gave an identical CD spectrum to that of wild-type GDH; t he spectrum of the double mutant was similar, although somewhat altere d in intensity. The results confirm the key role of K89 in dicarboxyla te recognition by GDH.