Selectivity in the binding of NAD(P)+ analogues to NAD- and NADP-dependent pig heart isocitrate dehydrogenases. A nuclear magnetic resonance study.
The coenzyme selectivity of pig heart NAD-dependent and NADP-dependent isocitrate dehydrogenase has been investigated by nuclear magnetic resonance through the use of coenzyme analogues. For both isocitrate dehydrogenases, more than 10-fold lower maximal activity is observed with thionicotinamide adenine dinucleotide [sNAD(P)+] than with NAD(P)+ or acetylpyridine adenine dinucleotide [acNAD-(P)+] as coenzyme. Nuclear Overhauser effect measurements failed to reveal any differences in the adenine-ribose conformations among the enzyme-bound analogues. The 2'-phosphate resonance of the enzyme-bound NADP+ analogues showed the same change in chemical shift observed for the natural coenzyme and revealed the same lack of pH dependence in the range from pH 5.4 to 8.2. NADP-dependent isocitrate dehydrogenase exhibits only small differences in Michaelis constants for the coenzymes with various nicotinamide substituents, reflecting a predominant role for the adenosine moiety in binding. The conformation of the bound nicotinamide-ribose of the natural coenzymes was appreciably different from that of the coenzyme, sNAD(P)+, which shows low catalytic activity. For both isocitrate dehydrogenases, sNAD(P)+ bound to the enzymes exhibits a mixture of syn and anti conformations while only the anti conformation can be detected for NAD(P)+. Chemical shifts of NAD(P)+ enriched with 13C in the carboxamide indicate that interaction of this group with the enzymes may play a role in positioning the nicotinamide ring to participate in catalysis. Our results suggest that, although interaction of the nicotinamide moiety with the enzymes contributes relatively little to the energy of interaction in the binary complex, the enzymes must correctly position this group for the catalytic event.(ABSTRACT TRUNCATED AT 250 WORDS)
Ehrlich
R S
RS
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Colman
R F
RF
D000818:Animals; D006639:Histidine / Q000032:analysis; D007521:Isocitrate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D009206:Myocardium / Q000201:enzymology*; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010758:Phosphorus; D013552:Swine
D009243:NAD; D010758:Phosphorus; D006639:Histidine; D009249:NADP; D007521:Isocitrate Dehydrogenase