Ligands of the Mn2+ bound to porcine mitochondrial NADP-dependent isocitrate dehydrogenase, as assessed by mutagenesis.
Pig heart mitochondrial NADP-dependent isocitrate dehydrogenase requires a divalent metal ion for catalysis, and metal-isocitrate is its preferred substrate. On the basis of the crystal structure of the enzyme-Mn(2+)-isocitrate complex, Asp(252), Asp(275), and Asp(279) were selected as targets for site-directed mutagenesis to evaluate the roles of these residues as ligands of the metal ion. The circular dichroism spectra of the purified mutant enzymes are similar to that of wild-type enzyme indicating there are no appreciable conformational changes. The K(m) values for isocitrate and for Mn(2+) are increased in the asparagine and histidine mutants at positions 252 and 275; while for cysteine mutants at the same positions, the K(m)'s are not changed appreciably. Mutants at position 279 exhibit only a small change in K(m) for isocitrate. These results indicate that Asp(252) and Asp(275) are ligands of enzyme-bound Mn(2+)and influence the binding of Mn(2+)-isocitrate. Cysteine is an acceptable substitute for aspartate as a ligand of Mn(2+). The pK(aes)'s of D252C and D275C enzymes are similar to that of the wild-type enzyme (about 5.2), while the pK(aes) of D279C is a little lower (about 4.7). These findings suggest that the V(max)'s of the D252C, D275C, and D279C enzymes depend on the ionizable form of the same group as in wild-type enzyme and neither Asp(252), Asp(275), nor Asp(279) acts as the general base in the enzymatic reaction. For wild-type enzyme, the pK(aes) varies with the metal ion used with Mg(2+) > Cd(2+) > Mn(2+) > Co(2+), similar to the order of the pK's for these four metal-bound waters. We therefore attribute the pH dependence of V(max) to the deprotonation of the metal-coordinated hydroxyl group of isocitrate bound to isocitrate dehydrogenase.
Huang
Yu Chu
YC
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19176, USA.
Grodsky
Neil B
NB
Kim
Tae-Kang
TK
Colman
Roberta F
RF
D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002413:Cations, Divalent / Q000737:chemistry; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D007521:Isocitrate Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D007700:Kinetics; D008024:Ligands; D008345:Manganese / Q000737:chemistry*; D008929:Mitochondria, Heart / Q000201:enzymology* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013379:Substrate Specificity / Q000235:genetics; D013552:Swine
D002413:Cations, Divalent; D008024:Ligands; D011994:Recombinant Proteins; D001224:Aspartic Acid; D008345:Manganese; D007521:Isocitrate Dehydrogenase; C039355:isocitrate dehydrogenase (NADP+)