ENGINEERING ACTIVITY AND STABILITY OF THERMOTOGA-MARITIMA GLUTAMATE-DEHYDROGENASE - I - INTRODUCTION OF A 6-RESIDUE ION-PAIR NETWORK IN THEHINGE REGION

Comparison of the recently determined three-dimensional structures of several glutamate dehydrogenases allowed for the identification of a f ive-residue ion-pair network in the hinge region of Pyrococcus furiosu s glutamate dehydrogenase (melting temperature 113 degrees C), that is not present in the homologous glutamate dehydrogenase from Thermotoga maritima (melting temperature 93 degrees C). In order to study the ro le of this ion-pair network, we introduced it into the T. maritima enz yme using a site-directed mutagenesis approach. The resulting T. marit ima glutamate dehydrogenases N97D, G376 K and N97D/G376 K as well as t he wild-type enzyme were overproduced in Escherichia coli and subseque ntly purified. Elucidation of the three-dimensional structure of the d ouble mutant N97D/G376 K at 3.0 Angstrom, showed that the designed ion -pair interactions were indeed formed. Moreover, because of interactio ns with an additional charged residue, a six-residue network is presen t in this double mutant. Melting temperatures of the mutant enzymes N9 7D, G376 K and N97D/G376 K, as determined by differential scanning cal orimetry, did not differ significantly from that of the wild-type enzy me. Identical transition midpoints in guanidinium chloride-induced den aturation experiments were found for the wild-type and all mutant enzy mes. Thermal inactivation at 85 degrees C occured more than twofold fa ster for all mutant enzymes than for the wild-type glutamate dehydroge nase. At temperatures of 65 degrees C and higher, the wild-type and th e three mutant enzymes showed identical specific activities. However, at 58 degrees C the specific activity of N97D/G376 K and G376 K was fo und to be significantly higher than that of the wildtype and N97D enzy mes. These results suggest that the engineered ion-pair interactions i n the hinge region do not affect the stability towards temperature or guanidinium chloride-induced denaturation but rather affect the specif ic activity of the enzyme and the temperature at which it functions op timally. (C) 1998 Academic Press.