Histidine in the Nucleotide‐Binding Site of NADP‐Linked Isocitrate Dehydrogenase from Pig Heart

Ehrlich, Robert S.; Colman, Roberta F.

doi:10.1111/j.1432-1033.1978.tb12562.x

Cited by 28 publications

(30 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our results also showed the absence of an essential reactive cysteine residue at the active site of this enzyme. The second‐order rate constant ( k 2 ) for inactivation of the enzyme by DEPC, 3 m −1 ·s −1 (180 m −1 · min −1 ) at pH 6.5 is within the range of the values obtained for the reaction of DEPC with histidine residues of different enzymes (10–1800 m −1 ·min −1 at pH 6 and 25 °C) or with the imidazole ring in model compounds (150–200 m −1 ·min −1 ) [37,40]. For pig‐heart NADP‐isocitrate dehydrogenase the value of this constant is 200 m −1 ·min −1 at pH 6.0.…”

Section: Discussionsupporting

confidence: 68%

“…In the presence of 5 m urea (data not shown), the number of total histidine residues/subunit of C. acremonium NADP‐isocitrate dehydrogenase was estimated to be eight. For the pig‐heart enzyme, Ehrlich and Colman [37] reported a total number of 14 histidine residues modified by DEPC.…”

Section: Resultsmentioning

confidence: 99%

“…For pig‐heart NADP‐isocitrate dehydrogenase the value of this constant is 200 m −1 ·min −1 at pH 6.0. The p K a value of 6.9 resembles the characteristic values of the histidine residues modified in other proteins [29,31,36,37,41,42].…”

Section: Discussionmentioning

confidence: 98%

“…The fact that NADP and NADPH partially protected the C. acremonium enzyme against DEPC inactivation suggests an indirect interaction of the histidine residue with the coenzyme binding site. The pig enzyme is inactivated by modification of histidine by DEPC, with protection being afforded by NADP and NADPH, although the modified histidine residue was not identified [37]. From X‐ray crystallographic studies, His339 has been located near the adenine portion of the coenzyme site in the E. coli enzyme [12].…”

Section: Discussionmentioning

confidence: 99%

See 3 more Smart Citations

Chemical modification of NADP‐isocitrate dehydrogenase from Cephalosporium acremonium

Olano

Soler

Busto

et al. 1999

European Journal of Biochemistry

View full text Add to dashboard Cite

NADP-isocitrate dehydrogenase from Cephalosporium acremonium CW-19 has been inactivated by diethyl pyrocarbonate following a first-order process giving a second-order rate constant of 3.0 m 21´s21 at pH 6.5 and 25 8C. The pH-inactivation rate data indicated the participation of a group with a pK value of 6.9. Quantifying the increase in absorbance at 240 nm showed that six histidine residues per subunit were modified during total inactivation, only one of which was essential for catalysis, and substrate protection analysis would seem to indicate its location at the substrate binding site. The enzyme was not inactivated by 5,5 H -dithiobis(2-nitrobenzoate), N-ethylmaleimide or iodoacetate, which would point to the absence of an essential reactive cysteine residue at the active site. Pyridoxal 5 H -phosphate reversibly inactivated the enzyme at pH 7.7 and 5 8C, with enzyme activity declining to an equilibrium value within 15 min. The remaining activity depended on the modifier concentration up to about 2 mm. The kinetic analysis of inactivation and reactivation rate data is consistent with a reversible two-step inactivation mechanism with formation of a noncovalent enzyme-pyridoxal 5 H -phosphate complex prior to Schiff base formation with a probable lysyl residue of the enzyme. The analysis of substrate protection shows the essential residue(s) to be at the active site of the enzyme and probably to be involved in catalysis.Keywords: NADP-isocitrate dehydrogenase; chemical modification; Cephalosporium acremonium.Although the NADP + -dependent isocitrate dehydrogenases from pig heart, yeast and Escherichia coli have been extensively studied (reviewed in [1,2]), the complete metabolic functions and several structural features remain to be established. Cephalosporium acremonium produces the b-lactam antibiotic cephalosporin C. The condensation of l-cysteine with l-aaminoadipic acid is the first step in the biosynthetic pathway of all b-lactam antibiotics, where 2-oxoglutarate is not only a precursor of l-a-aminoadipic acid [3], but it is also required in two other steps, those catalyzed by the deacetoxycephalosporin C synthetase and by the deacetoxycephalosporin C hydrolase [4±6]. In C. acremonium, the availability of 2-oxoglutarate has been proposed [7,8] as a limiting factor for cephalosporin C biosynthesis. Due to the potential role of the isocitrate dehydrogenase [threo-d s -isocitrate NADP + oxidoreductase (decarboxylating)] from C. acremonium in supplying 2-oxoglutarate for cephalosporin C biosynthesis, we decided to investigate some regulatory properties of this enzyme. We have shown that it is a dimer of identical subunits, is not allosterically regulated and is unusually stable to temperature in the presence of its true substrate, the magnesium-isocitrate complex [9]. Furthermore, we found maximum values of total NADP-isocitrate dehydrogenase activity in the 4±5 day range on fermentation medium [9], at the time of maximum value of deacetoxycephalosporin C synthetase [10].Knowledge of the role of individual a...

show abstract

Section: Discussionsupporting

confidence: 68%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

Chemical modification of NADP‐isocitrate dehydrogenase from Cephalosporium acremonium

Olano

Soler

Busto

et al. 1999

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…The technique of affinity labeling, using nucleotide analogues with reactive functional groups, can potentially result in more tThis work was supported by USPHS Grant DK 39075. specific chemical modification and should allow the identification of critical amino acid residues within the nucleotide binding site (Colman, 1983b). Studies of the binding of coenzymes and coenzyme fragments to NADP+-specific isocitrate dehydrogenase have demonstrated that a 2,-phosphate is essential for the enzyme-nucleotide interaction (Ehrlich & Colman, 1978;Mas & Colman, 1985), suggesting that any potential affinity label for this enzyme retain the 2'-phosphate.…”

mentioning

confidence: 99%

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

Bailey¹,

Colman²

1987

Biochemistry

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Modeling substrate binding in Thermus thermophilus isopropylmalate dehydrogenase

Zhang

Koshland

1995

Protein Science

View full text Add to dashboard Cite

The Thermus thermophilus 3-isopropylmalate dehydrogenase (IPMDH) and Escherichia coli isocitrate dehydrogenase (ICDH) are two functionally and evolutionarily related enzymes with distinct substrate specificities. To understand the determinants of substrate specificities of the two proteins, the substrate and coenzyme in IPMDH were docked into their respective binding sites based on the published structure for apo IPMDH and its sequence and structural homology to ICDH. This modeling study suggests that (1) the substrate and coenzyme (NAD) binding modes of IPMDH are significantly different from those of ICDH, (2) the interactions between the substrates and coenzymes help explain the differences in substrate specificities of IPMDH and ICDH, and (3) binding of the substrate and coenzyme should induce a conformational change in the structure of IPMDH.Keywords: docking; isocitrate dehydrogenase; isopropylmalate dehydrogenase; substrate binding Thermus thermophilus3-isopropylmalate dehydrogenase (IPMDH) and Escherichia coli isocitrate dehydrogenase (ICDH) are two closely related enzymes that catalyze similar reactions in different metabolic pathways. IPMDH functions in the leucine biosynthesis pathway and catalyzes the oxidative decarboxylation of isopropylmalate to a-ketoisocaproate (Imada et al., 1991). ICDH catalyzes a similar decarboxylation reaction in the carbohydrate metabolism (Kornberg, 1966), and converts isocitrate to a-ketoglutarate. The two reactions are shown in Figure IA. Both reactions are postulated to proceed in two steps, with dehydrogenation preceding decarboxylation (Fig. IB) (Grissom & Cleland, 1985; Pirrung et ai., 1994).Most of the postulated key catalytic residues in E. coli ICDH (Hurley et ai., 1991) are conserved in 7: thermophilus lPMDH (Imada et al., 1991;Miyazaki et al., 1992). The two proteins have an overall 30% sequence identity (Fig. 2). The conservation of catalytic residues also extends to the IPMDHs and ICDHs of other species (Fig. 2), suggesting that these proteins may have a common evolutionary origin and structural, functional properties.The substrate specificities of 7: thermophilus lPMDH and E. coli ICDH are, however, very different. ICDH shows no detectable activities for isopropylmalate and IPMDH shows no activity for isocitrate (Miyazaki et ai., 1993). A mammalian form of ICDH has also been extensively investigated (Colman, 1973 , 1978). showed that 7: thermophilus IPMDH has broad substrate specificities against alkyl-malates. Substituting the y-moiety of isopropylmalate with methyl, ethyl, or propyl groups has little effect on the overall catalytic efficiency (kcor/K,,), suggesting that the hydrophobic packing between the substrate and the enzyme-coenzyme complex is not very specific. On the other hand, IPMDH shows no detectable activity against isocitrate, suggesting that the polar or charge-charge interaction with the y-carboxyl group destabilizes the IPMDH-substrate-coenzyme complex . Studies on the roles of catalytic residues of IPMDH are limited. It was shown...

show abstract

Histidine in the Nucleotide‐Binding Site of NADP‐Linked Isocitrate Dehydrogenase from Pig Heart

Cited by 28 publications

References 31 publications

Chemical modification of NADP‐isocitrate dehydrogenase from Cephalosporium acremonium

Chemical modification of NADP‐isocitrate dehydrogenase from Cephalosporium acremonium

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

Modeling substrate binding in Thermus thermophilus isopropylmalate dehydrogenase

Contact Info

Product

Resources

About