Affinity labeling of nicotinamide adenine dinucleotide-dependent isocitrate dehydrogenase by the 2',3'-dialdehyde derivative of adenosine 5'-diphosphate. Evidence for the formation of an unusual reaction product

King, Michael M.; Colman, Roberta F.

doi:10.1021/bi00276a021

Cited by 43 publications

(33 citation statements)

References 32 publications

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“…Neither the presence of NaBHXN (5 mM), a selective reducing agent of Schiff bases [21], during incubation with the oAdN, nor the addition of NaBH4 (10 mM) after incubation, was necessary for inactivation of ATPase. This was also shown for the inactivation of pyruvate kinase and NADisocitrate dehydrogenase by oAdN [22,23], and beef heart ATPase [lo]. All our experiments were carried out at pH 8.0 since inactivation was about 3-times faster at this pH than at neutral pH.…”

Section: Kinetics Of Inactivation Of Fl By Oadnsupporting

confidence: 65%

Inactivation of beef heart mitochondrial F₁‐ATPase by the 2′,3′‐dialdehyde derivatives of adenine nucleotides

1984

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Beef fietzrf mitochondri~~ Ft-ATPase was i~~~t~vat~ by the 2" ,3 '-dialdehyde derivatives of ATP, ADP and AMP {ATP, oADP, oAMP). In the absence of Mg2+, i~a~t~~~on resuIWl from the binding Of 1 mol nuekotide analog per active unit of Fi. The most efficient andog was oADP, followed by oAMP and oATP, Complete inactivsxtion was correlated with the binding; of about 11 moi ["%]oADPr'mol Ft, After ~orr~~ti~r~ for non-specifk labeling, the ~~~~ber of sp~ifi~~~ly bound fi4C]oADP was 2-3 mol per mol Fi. By S~S-polyacryl~m~~~ gel eiectrophoresis, [i4C]oADP was found to bind covalently mainly to the LY and fl subunits. In the presence of Mga', oATP behaved 8s a substrate and was sfowly hydrolyzed.~ucl~~de sites in the Ft sector of m~tocb~n-

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Section: Kinetics Of Inactivation Of Fl By Oadnsupporting

confidence: 65%

Inactivation of beef heart mitochondrial F₁‐ATPase by the 2′,3′‐dialdehyde derivatives of adenine nucleotides

1984

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“…The oxidation of nucleotides by periodate yields 2′,3′‐dialdehyde derivatives that are capable of reacting with lysine residues [21,24]. The present studies showed that the 2′,3′‐dialdehyde derivative of ADP reacts specifically with C. boidinii formate dehydrogenase exhibiting the characteristics of an affinity label for the nucleotide binding site of the enzyme [21,24,35–40]. The inactivation of FDH by oADP obeyed saturation biphasic kinetics that indicates that the oADP forms a dissociable complex with the enzyme prior to covalent modification [22–24,34].…”

Section: Discussionmentioning

confidence: 99%

Characterization of the NAD⁺ binding site of Candida boidinii formate dehydrogenase by affinity labelling and site‐directed mutagenesis

Labrou¹,

Rigden²,

Clonis³

2000

European Journal of Biochemistry

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The 2 H ,3 H -dialdehyde derivative of ADP (oADP) has been shown to be an affinity label for the NAD 1 binding site of recombinant Candida boidinii formate dehydrogenase (FDH). Inactivation of FDH by oADP at pH 7.6 followed biphasic pseudo first-order saturation kinetics. The rate of inactivation exhibited a nonlinear dependence on the concentration of oADP, which can be described by reversible binding of reagent to the enzyme (K d 0.46 mm for the fast phase, 0.45 mm for the slow phase) prior to the irreversible reaction, with maximum rate constants of 0.012 and 0.007 min 21 for the fast and slow phases, respectively. Inactivation of formate dehydrogenase by oADP resulted in the formation of an enzyme±oADP product, a process that was reversed after dialysis or after treatment with 2-mercaptoethanol (. 90% reactivation). The reactivation of the enzyme by 2-mercaptoethanol was prevented if the enzyme±oADP complex was previously reduced by NaBH 4 , suggesting that the reaction product was a stable Schiff's base. Protection from inactivation was afforded by nucleotides (NAD 1 , NADH and ADP) demonstrating the specificity of the reaction. When the enzyme was completely inactivated, approximately 1 mol of [ 14 C]oADP per mol of subunit was incorporated. Cleavage of [ 14 C]oADPmodified enzyme with trypsin and subsequent separation of peptides by RP-HPLC gave only one radioactive peak. Amino-acid sequencing of the radioactive tryptic peptide revealed the target site of oADP reaction to be Lys360. These results indicate that oADP inactivates FDH by specific reaction at the nucleotide binding site, with negative cooperativity between subunits accounting for the appearance of two phases of inactivation. Molecular modelling studies were used to create a model of C. boidinii FDH, based on the known structure of the Pseudomonas enzyme, using the modeller 4 program. The model confirmed that Lys360 is positioned at the NAD 1 -binding site. Site-directed mutagenesis was used in dissecting the structure and functional role of Lys360. The mutant Lys3603Ala enzyme exhibited unchanged k cat and K m values for formate but showed reduced affinity for NAD 1 . The molecular model was used to help interpret these biochemical data concerning the Lys3603Ala enzyme. The data are discussed in terms of engineering coenzyme specificity.

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“…Clearly, oCTP and not contaminating anhydro-oC are responsible for most of the inactivation. Others have demonstrated this type of enzyme accelerated b-elimination reaction with radioactively labeled dialdehyde nucleotides~Lowe & Beechey, 1982; King & Colman, 1983!. Some studies using periodate oxidized nucleotides have provided evidence that the covalent modification proceeds by formation of a Schiff base while others have indicated that a morpholino structure is formed~Hansske et al, 1974; Gregory & Kaiser, 1979;Lowe & Beechey, 1982;King & Colman, 1983, 1990Mas & Colman, 1983;Rayford et al, 1985;White & Levy, 1987;Colman, 1990!. Morpholino derivatives are fairly stable under moderate conditions, but harsher treatment releases or degrades the modifi- peaks at m0z 1,759.8 and 1,761.8 with a single major prompt fragment at m0z 1,650.7.…”

Section: Discussionmentioning

confidence: 99%

Covalent modification of lys19 in the ctp binding site of cytidine 5′‐monophosphate n‐acetylneuraminic acid synthetase

1999

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Periodate oxidized CTP~oCTP! was used to investigate the importance of lysine residues in the CTP binding site of the cytidine 59-monophosphate N-acetylneuraminic acid~CMP-NeuAc! synthetase~EC 2.7.7.43! from Haemophilus ducreyi. The reaction of oCTP with the enzyme follows pseudo-first-order saturation kinetics, giving a maximum rate of inactivation of 0.6 min Ϫ1 and a K I of 6.0 mM at pH 7.1. Mass spectrometric analysis of the modified enzyme provided data that was consistent with b-elimination of triphosphate after the reaction of oCTP with the enzyme. A fully reduced enzyme-oCTP conjugate, retaining the triphosphate moiety, was obtained by inclusion of NaBH 3 CN in the reaction solution. The b-elimination product of oCTP reacted several times more rapidly with the enzyme compared to equivalent concentrations of oCTP. This compound also formed a stable reduced morpholino adduct with CMP-NeuAc synthetase when the reaction was conducted in the presence of NaBH 3 CN, and was found to be a useful lysine modifying reagent. The substrate CTP was capable of protecting the enzyme to a large degree from inactivation by oCTP and its b-elimination product. Lys19, a residue conserved in CMP-NeuAc synthetases, was identified as being labeled with the b-elimination product of oCTP.Keywords: affinity labeling; CMP-NeuAc synthetase; Haemophilus ducreyi; mass spectrometry; periodate oxidized CTP; sialic acid Sialic acids are nine carbon a-keto acid monosaccharides that have many important biological effects, particularly in cellular recognition~Schauer, 1985;Kelm et al., 1996!. Although commonly found in higher animals and present in all mammals, sialic acids have been found in relatively few microorganisms~Schauer et al., 1995!. In bacteria, sialic acid presents as the terminal residue of lipooligosaccharides~LOS! and in the form of polymeric extracellular capsules is capable of masking surface epitopes and inhibiting phagocytosis of the bacteria~Timmis et al., 1985;Parsons et al., 1988;Rest & Frangipane, 1992!. Haemophilus ducreyi is the Gram-negative bacterium that is the causative agent of the sexually transmitted disease chancroid. Chancroid is common in Africa, Asia, and Latin America. Although the incidence of cases in the United States is low, outbreaks have occurred in recent years~Trees & Morse, 1995!. In addition, the incidence of the disease may be greater than previously estimated Webb et al., 1995!. The LOS of H. ducreyi has been determined to be a virulence factor~Odumeru et al., 1987;Campagnari et al., 1991;Lagergard, 1992!. Furthermore, the LOS from H. ducreyi, along with other Haemophilus and Neisseria spp., are structurally similar to the carbohydrates of some human glycolipids and glycoproteins~Campagnari et al ., 1990;Mandrell & Apicella, 1993!. It has been hypothesized that this mimicry may allow the organism to evade host immune defenses and utilize host receptors for adherence. Sialic acid has recently been identified as a major component of the LOS from many strains of H. ducreyi, yet the preci...

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Affinity labeling of nicotinamide adenine dinucleotide-dependent isocitrate dehydrogenase by the 2',3'-dialdehyde derivative of adenosine 5'-diphosphate. Evidence for the formation of an unusual reaction product

Cited by 43 publications

References 32 publications

Inactivation of beef heart mitochondrial F₁‐ATPase by the 2′,3′‐dialdehyde derivatives of adenine nucleotides

Inactivation of beef heart mitochondrial F₁‐ATPase by the 2′,3′‐dialdehyde derivatives of adenine nucleotides

Characterization of the NAD⁺ binding site of Candida boidinii formate dehydrogenase by affinity labelling and site‐directed mutagenesis

Covalent modification of lys19 in the ctp binding site of cytidine 5′‐monophosphate n‐acetylneuraminic acid synthetase

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Affinity labeling of nicotinamide adenine dinucleotide-dependent isocitrate dehydrogenase by the 2',3'-dialdehyde derivative of adenosine 5'-diphosphate. Evidence for the formation of an unusual reaction product

Cited by 43 publications

References 32 publications

Inactivation of beef heart mitochondrial F1‐ATPase by the 2′,3′‐dialdehyde derivatives of adenine nucleotides

Inactivation of beef heart mitochondrial F1‐ATPase by the 2′,3′‐dialdehyde derivatives of adenine nucleotides

Characterization of the NAD+ binding site of Candida boidinii formate dehydrogenase by affinity labelling and site‐directed mutagenesis

Covalent modification of lys19 in the ctp binding site of cytidine 5′‐monophosphate n‐acetylneuraminic acid synthetase

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Inactivation of beef heart mitochondrial F₁‐ATPase by the 2′,3′‐dialdehyde derivatives of adenine nucleotides

Inactivation of beef heart mitochondrial F₁‐ATPase by the 2′,3′‐dialdehyde derivatives of adenine nucleotides

Characterization of the NAD⁺ binding site of Candida boidinii formate dehydrogenase by affinity labelling and site‐directed mutagenesis