Cosubstrate and allosteric modifier activities of structural analogues of NAD and ADP for NAD-specific isocitrate dehydrogenase from bovine heart

Plaut, G.W.; Cheung, C.P.; Suhadolnik, R.J.; Aogaichi, T.

Biochemistry 18(15): 3430-3438


ISSN/ISBN: 0006-2960
PMID: 223632
DOI: 10.1021/bi00582a034
Accession: 068524679

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The specificity of bovine heart NAD-linked isocitrate dehydrogenase (EC 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 1,N6-ethenoadenosine were inactive, but the formycin analogue had cosubstrate activity. Values of Km for 2'-dNAD+ and 3'-dNAD+ were about 5- to 7-fold larger than for NAD+; Vmax was about the same for 2'-NAD+ and NAD+, and Vmax was about 1/5 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 activitation 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. .alpha.-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 H at C-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. N-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 N-9 and C-8 of ADP are replaced by C and N, 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-.alpha.-S and ADP-.beta.-S were equivalent to ADP as positive effectors. However, activity was lost when the .beta.-phosphate group was replaced by sulfate (adenosine 5'-phosphosulfate) or when the bridge oxygen between the .alpha.- and .beta.- or .beta.- and .gamma.-phosphates was replaced by a methylene group (.alpha.,.beta.-methylene-ADP or .beta.-.gamma.-methylene-ATP).