COSUBSTRATE AND ALLOSTERIC MODIFIER ACTIVITIES OF STRUCTURAL ANALOGS OF NAD AND ADP FOR NAD-SPECIFIC ISOCITRATE DEHYDROGENASE FROM BOVINE HEART

The specificity of bovine heart NAD-linked isocitrate dehydrogenase for the configurations of cosubstrate (NAD+) and allosteric effector (ADP) was examined with 5 NAD+ analogues modified in the adenosine portion and over 20 analogues of ADP altered in the purine ring, pentosyl group, and 5′-pyrophosphate group. NAD analogues in which the adenosine portion was replaced by inosine or l, N6-ethenoadenosine were inactive, but the formycin analogue had cosubstrate activity. Values of Km for 2′-dNAD+ and 3′-dNAD+ were about five- to sevenfold larger than for NAD+; Kmax was about the same for 2′-dNAD+ and NAD+, and Vmax was about one-fifth for 3′-dNAD+ compared with NAD+. The configuration or nature of substituents about carbons 2′ and 3′ of the ribosyl portion of ADP is not critical for allosteric activation since ADP analogues containing 2′-deoxy-d-ribose, 3′-deoxy-d-ribose, 2′, 3′-dideoxy-d-ribose, 2′-O-methyl-d-ribose, and d-arabinose were about equally effective. α-ADP, where the glycosidic linkage is inverted, inhibits activation by ADP competitively. An unsubstituted 6-amino group and nitrogen 1 in ADP are essential for activity since N6, N6-dimethylaminopurine ribonucleoside 5′-pyrophosphate, 1-N6-etheno-ADP, IDP, and 1-N-oxide-ADP were neither activators nor inhibitors. A hydrogen at carbon 2 of ADP is not essential for modifier activity since 2, 6-diaminopurine nucleoside diphosphate was an activator; however, 2-hydroxy-6-aminopurine nucleoside diphosphate was inactive. Nitrogen 7 of ADP is not required for activity since the tubercidin analogue was as effective as ADP. The capability to bind to the allosteric site is retained when nitrogen 9 and carbon 8 of ADP are replaced by carbon and nitrogen, respectively, since the formycin analogue (FDP) was an inhibitor competitive with ADP. 8-Br-ADP was inactive. The secondary phosphates of the 5′-pyrophosphoryl group of ADP are required for activity since ADP-amide (P1-(adenosine-5′)-P2-aminopyrophosphate) was a positive modifier and 5′-adenylyl imidodiphosphate was an inhibitor competitive with ADP. Oxygen functions at the pyrophosphate group can be replaced by sulfur since ADP-α-S and ADP-β-S were equivalent to ADP as positive effectors. However, activity was lost when the β-phosphate group was replaced by sulfate (adenosine 5′-phosphosulfate) or when the bridge oxygen between the α- and β- or β- and 7-phosphates was replaced by a methylene group (α, β-methylene-ADP or β, γ-methylene- ATP). © 1979, American Chemical Society. All rights reserved.