Histidine 454 plays an important role in polymerization of human glutamate dehydrogenase.
Although previous chemical modification studies have suggested several residues to be involved in the maintenance of the quaternary structure of glutamate dehydrogenase (GDH), there are conflicting views for the polymerization process and no clear evidence has been reported yet. In the present study, cassette mutagenesis at seven putative positions (Lys333, Lys337, Lys344, Lys346, Ser445, Gly446, and His454) was performed using a synthetic human GDH gene to examine the polymerization process. Of the mutations at the seven different sites, only the mutagenesis at His454 results in depolymerization of the hexameric GDH into active trimers as determined by HPLC gel filtration analysis and native gradient polyacrylamide gel electrophoresis. The mutagenesis at His454 has no effects on expression or stability of the protein. The K(M) values for NADH and 2-oxoglutarate were 1.5-fold and 2.5-fold greater, respectively, for the mutant GDH than for wild-type GDH, indicating that substitution at position 454 had appreciable effects on the affinity of the enzyme for both NADH and 2-oxoglutarate. The V(max) values were similar for wild-type and mutant GDH. The k(cat)/K(M) value of the mutant GDH was reduced up to 2.8-fold. The decreased efficiency of the mutant, therefore, results from the increase in K(M) values for NADH and 2-oxoglutarate. The results with cassette mutagenesis and HPLC gel filtration analysis suggest that His454 is involved in the polymerization process of human GDH.
Lee
Eun-Young
EY
Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul, South Korea.
Huh
Jae-Wan
JW
Yang
Seung-Ju
SJ
Choi
Soo Young
SY
Cho
Sung-Woo
SW
Choi
Hyun Jin
HJ
D001704:Biopolymers / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D016254:Mutagenesis, Insertional
D001704:Biopolymers; D006639:Histidine; D005969:Glutamate Dehydrogenase