Each conserved active site tyr in the three subunits of human isocitrate dehydrogenase has a different function

Abstract

The human NAD-dependent isocitrate dehydrogenase (IDH) is a heterotetrameric mitochondrial enzyme with 2alpha:1beta:1gamma subunit ratio. The three subunits share 40-52% identity in amino acid sequence and each includes a tyrosine in a comparable position: alphaY126, betaY137, and gammaY135. To study the role of the corresponding tyrosines of each of the subunits of human NAD-IDH, the tyrosines were mutated (one subunit at a time) to Ser, Phe, or Glu. Enzymes were expressed with one mutant and two wild-type subunits. The results of characterization of the mutant enzymes suggest that betaY137 is involved in NAD binding and allosteric activation by ADP. The alphaY126 is required for catalytic activity and likely acts as a general acid in the reaction. The gammaY135 is also required for catalytic activity and may be involved in proper folding of the enzyme. The corresponding tyrosines in the three dissimilar subunits of NAD-IDH thus have distinctive functions.

FIGURE 1.

Comparison of the amino acid sequences of the E. coli NADP-specific isocitrate dehydrogenase (E. coli-IDH) with the α subunit (α-NAD-IDH), β subunit (β-NAD-IDH), and the γ subunit (γ-NAD-IDH) of human NAD-dependent isocitrate dehydrogenase. A, complete sequence alignment; B, partial alignment highlighting the conserved Tyr and including the pig NADP-dependent isocitrate dehydrogenase (Pig NADP-IDH). The sequence alignment was performed for the E. coli NADP-IDH and all three subunits of human NAD-IDH using ClustalW. The (*) indicates that the amino acids at the position are identical, (:) indicates they are closely similar, and (.) indicates they are similar. The pig NADP-IDH was included in the alignment manually.

FIGURE 2.

SDS-PAGE of recombinant wild-type and mutant human NAD-dependent isocitrate dehydrogenase. Lane A, marker; lane B, wild-type; lane C, αY126S; lane D, αY126F; lane E, αY126E; lane F, βY137S; lane G, βY137F; lane H, βY137E.

FIGURE 3.

Western blot comparison of the wild-type and γY135F mutant human NAD-IDH. The protein concentrations of the cell lysate supernatants were 20.6 mg/ml and 18.8 mg/ml for the wild-type and γY135F mutant, respectively. The areas of the band stained with antibody against the γ subunit are very similar.

FIGURE 4.

CD spectra of purified human recombinant wild-type and mutant NAD-IDH enzymes: Wild-type (●), αY126S (○), αY126F (▾),αY126E (▿), βY137S (■), βY137F (□), βY137E (♦) in buffer containing 25 mm triethanolamine chloride buffer, pH 7. 4, containing 10% glycerol, 0.2 mm MnSO₄.

FIGURE 5.

pH profile of wild-type and mutant enzymes. A, wild-type and β subunit mutants: Wild-type (▴); βY137S (●); βY137F (○); βY137E (▾) in the following buffers: sodium acetate (pH 4.4–5.8), MES (pH 5.4–6.6), and PIPES (pH 6.2–7.4), and triethanolamine hydrochloride (pH 6.8–7.6) at standard substrate concentrations. B, α subunit mutants: αY126S (♦); αY126F (⎔); αY126E (▵). The buffers used were same as in A, except 5 mm NAD was used to assay the αY126E mutant enzyme.