2-[(4-Bromo-2,3-dioxobutyl)thio]- and 2-[(3-bromo-2-oxopropyl)thio]adenosine 2',5'-bisphosphate: new nucleotide analogs that act as affinity labels of nicotinamide adenine dinucleotide phosphate specific isocitrate dehydrogenase

Abstract

Two new reactive adenine nucleotide analogues have been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 2',5'-bisphosphate (2-BDB-TA-2',5'-DP) and 2-[(3-bromo-2-oxopropyl)thio]adenosine 2',5'-bisphosphate (2-BOP-TA-2',5'-DP). Starting with NADP+, 2'-phospho-adenosine 5'-(diphosphoribose) (PADPR) was generated enzymatically and was converted to PADPR 1-oxide by reaction with m-chloroperoxybenzoic acid. Treatment with NaOH followed by reaction with carbon disulfide yielded 2-thioadenosine 2',5'-bisphosphate (TA-2',5'-DP). Condensation of TA-2',5'-DP with 1,4-dibromobutanedione or 1,3-dibromo-2-propanone gave the final products 2-BDB-TA-2',5'-DP and 2-BOP-TA-2',5'-DP, respectively. The structure of these new reagents was determined by UV, 1H NMR, 31P NMR, and 13C NMR spectroscopy as well as by bromide and phosphorus analysis. Both of these reagents exhibit properties expected for an affinity label of the coenzyme site of NADP+-dependent isocitrate dehydrogenase. With both reagents, biphasic kinetics of inactivation are observed that can be described in terms of a fast initial phase of inactivation resulting in partially active enzyme of 6-7% residual activity, followed by a slower phase leading to total inactivation. The inactivation rate constants for both reagents exhibit a nonlinear dependence on reagent concentration, consistent with the formation of a reversible complex with the enzyme prior to irreversible modification. The enzyme incorporates both reagents to a limited extent and is protected against inactivation by NADP+ and NADPH. The reaction of these new nucleotide analogues with isocitrate dehydrogenase is compared to the much slower inactivation caused by bromoacetone, indicating the importance of the nucleotide moiety in the functioning of the affinity labels. It is likely that 2-BDB-TA-2',5'-DP and 2-BOP-TA-2',5'-DP will have general applicability as affinity labels for other NADP+ binding enzymes.

Adenosine 2‘-Monophosphate, 5‘-O-[S-(4-Succinimidylbenzophenone)- thiophosphate]: A New Photoaffinity Label for the Coenzyme Site of Porcine NADP-Specific Isocitrate Dehydrogenase

“…2′-P-AMPS-Succ-BP-modified C269A/C379S enzyme is capable of binding 0.41 mol of D-isocitrate/mol enzyme subunit and 0.51 mol of NADPH/ mol enzyme subunit (or 0.82 mol of D-isocitrate/mol enzyme dimer and 1.02 mol of NADPH/mol enzyme dimer). These ligand binding results for the 2′-P-AMPS-Succ-BP-modified C269A/C379S enzyme are similar to those of wild-type enzyme modified with 2-BDB-TA-2′,5′-DP or 2-BDB-T A-2′,5′-DP (25,26) and implies that 2′-P-AMPS-Succ-BP occupies only one of the two active sites of isocitrate dehydrogenase dimer.…”

“…The coenzyme site of porcine mitochondrial isocitrate dehydrogenase has been studied previously using the affinity labels, 2-(4-bromo-2,3-dioxobutylthio)-1, N 6 2′,5′-DP) (10,25,26,31). Each of these affinity labels is an adenosine analogue with a reactive bromoketo group linked to the C2 position of adenine and is capable of revealing active site residues in the vicinity of the nicotinamide ring of NADP.…”

Cysteinyl peptides of rabbit muscle pyruvate kinase labeled by the affinity label 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate

“…interpretation of 8-BDB-TA-5'-TP as a PEP analogue is the fact that 175 ;uM 8-[(3-bromo-2-oxopropyl)thio]-ATP (8-BOP-TA-5'-TP), which exists only in the keto form even in polar solvents (Bailey & Colman, 1987) and hence is "unlike" phosphoenolpyruvate, inactivates pyruvate kinase very slowly with a rate constant of 0.015 min'1 as compared with 0.23 min'1 for 8-BDB-TA-5'-TP under the same conditions (De-Camp, 1988). Similarly, the PEP analogue 2-BDB-T«A-5/-DP inactivates pyruvate kinase rapidly, while the same concentration of 2-BOP-TeA-5'-DP has little effect on activity (DeCamp & .…”

“…Similarly, the PEP analogue 2-BDB-T«A-5/-DP inactivates pyruvate kinase rapidly, while the same concentration of 2-BOP-TeA-5'-DP has little effect on activity (DeCamp & . In contrast, 2-[(4-bromo-2,3dioxobutyl)thio]adenosine 2',5'-bisphosphate and 2-[(3bromo-2-oxopropyl)thio]adenosine 2,,5,-bisphosphate, which function as affinity labels of the coenzyme site of NADP+dependent isocitrate dehydrogenase, inactivate that enzyme at comparable rates (Bailey & Colman, 1987).…”

Cysteinyl peptides labeled by dibromobutanedione in reaction with rabbit muscle pyruvate kinase

“…Bromodioxobutyl nucleotide derivatives have been shown to hydrolyze with the release of bromide. At pH 7 and 25 "C, the t1,2 for hydrolysis has been shown to be about 50 min (Bailey & Colman, 1987). For this reason, the rate constants were calculated from a least-squares fit to the experimental data over the first 30 min.…”