Evaluation by mutagenesis of the importance of 3 arginines in alpha, beta, and gamma subunits of human NAD-dependent isocitrate dehydrogenase.
Mammalian NAD-dependent isocitrate dehydrogenase is an allosteric enzyme, activated by ADP and composed of 3 distinct subunits in the ratio 2alpha:1beta:1gamma. Based on the crystal structure of NADP-dependent isocitrate dehydrogenases from Escherichia coli, Bacillus subtilis, and pig heart, and a comparison of their amino acid sequences, alpha-Arg88, beta-Arg99, and gamma-Arg97 of human NAD-dependent isocitrate dehydrogenase were chosen as candidates for mutagenesis to test their roles in catalytic activity and ADP activation. A plasmid harboring cDNA that encodes alpha, beta, and gamma subunits of the human isocitrate dehydrogenase (Kim, Y. O., Koh, H. J., Kim, S. H., Jo, S. H., Huh, J. W., Jeong, K. S., Lee, I. J., Song, B. J., and Huh, T. L. (1999) J. Biol. Chem. 274, 36866-36875) was used to express the enzyme in isocitrate dehydrogenase-deficient E. coli. Wild type (WT) and mutant enzymes (each containing 2 normal subunits plus a mutant subunit with alpha-R88Q, beta-R99Q, or gamma-R97Q) were purified to homogeneity yielding enzymes with 2alpha:1beta:1gamma subunit composition and a native molecular mass of 315 kDa. Specific activities of 22, 14, and 2 micromol of NADH/min/mg were measured, respectively, for WT, beta-R99Q, and gamma-R97Q enzymes. In contrast, mutant enzymes with normal beta and gamma subunits and alpha-R88Q mutant subunit has no detectable activity, demonstrating that, although beta-Arg99 and gamma-Arg97 contribute to activity, alpha-Arg88 is essential for catalysis. For WT enzyme, the Km for isocitrate is 2.2 mm, decreasing to 0.3 mm with added ADP. In contrast, for beta-R99Q and gamma-R97Q enzymes, the Km for isocitrate is the same in the absence or presence of ADP, although all the enzymes bind ADP. These results suggest that beta-Arg99 and gamma-Arg97 are needed for normal ADP activation. In addition, the gamma-R97Q enzyme has a Km for NAD 10 times that of WT enzyme. This study indicates that a normal alpha subunit is required for catalytic activity and alpha-Arg88 likely participates in the isocitrate site, whereas the beta and gamma subunits have roles in the nucleotide functions of this allosteric enzyme.
Soundar
Sambanthamurthy
S
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
Park
Jung-Hoon
JH
Huh
Tae-Lin
TL
Colman
Roberta F
RF
D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000495:Allosteric Site; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry*; D001412:Bacillus subtilis / Q000378:metabolism; D002384:Catalysis; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine; D013997:Time Factors
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D004247:DNA; D001120:Arginine; D007521:Isocitrate Dehydrogenase