Kinetics of Carboxypeptidase Action. I. Effect of Various Extrinsic Factors on Kinetic Parameters<sup>1</sup>

Group II intron self-splicing is essential for correct expression of organellar genes in plants, fungi, and yeast, as well as of bacterial genes. Self-excision of these autocatalytic introns from the primary RNA transcript is achieved in a two-step mechanism apparently analogous to the one of the eukaryotic spliceosome. The 2'-OH of a conserved adenosine (the branch point) located within domain 6 (D6) acts as the nucleophile in the first step of splicing. Despite the biological importance of group II introns, little is known about their structural organization and usage of metal ions in catalysis. Here we report the first solution structure of a catalytically active D6 construct encompassing the branch point and the neighboring helical regions from the mitochondrial yeast intron ai5 . The branch adenosine is the single unpaired nucleotide, and, in contrast to the spliceosomal branch site, resides within the helix being partially stacked between the two flanking GU wobble pairs. We identified a novel prominent Mg2+ binding site in the major groove of the branch site. Importantly, Mg2+ addition does not impair stacking of the branch-adenosine, but in contrast rather strengthens the interaction with the flanking uridines, as shown by NMR-and fluorescence studies. This means, that domain 6 presents the branch adenosine in a stacked fashion to the core of group II introns upon folding to the active conformation.

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“…To measure the pD value, 0.4 log units were added to the pH meter reading. [45,46] The RNA concentration was determined with a Varian…”

Section: Discussionmentioning

confidence: 99%

Solution Structure of Domain 6 from a Self‐Splicing Group II Intron Ribozyme: A Mg²⁺ Binding Site is Located Close to the Stacked Branch Adenosine

et al. 2007

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“…To measure the pD value, 0.4 log units were added to the pH meter reading. 40,41 The RNA concentration was determined by UV-VIS spectrophotometry, using an extinction coefficient at 260 nm (ε 260 ) of 296. in 3D as the solution structure of D6-27. 29 The branch-adenosine that is stacked within the helix is highlighted in red, the flanking GU-wobble pairs in gold.…”

Section: Nmr Sample Preparationmentioning

confidence: 99%

Metal ion-N7 coordination in a ribozyme branch domain by NMR

Erat¹,

Kovacs²,

Sigel³

2010

Journal of Inorganic Biochemistry

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The N7 of purine nucleotides presents one of the most dominant metal ion binding sites in nucleic acids. However, the interactions between kinetically labile metal ions like Mg2+ and these nitrogen atoms are inherently difficult to observe in large RNAs. Rather than using the insensitive direct N-15 detection, here we have used (2)J-H-1,N-15]-HSQC (Heteronuclear Single Quantum Coherence) NMR experiments as a fast and efficient method to specifically observe and characterize such interactions within larger RNA constructs. Using the 27 nucleotides long branch domain of the yeast-mitochondrial group II intron ribozyme Sc.ai5 gamma as an example, we show that direct N7 coordination of a Mg2+ ion takes place in a tetraloop nucleotide. A second Mg2+ ion, located in the major groove at the catalytic branch site, coordinates mainly in an outer-sphere fashion to the highly conserved flanking GU wobble pairs but not to N7 of the sandwiched branch adenosine. (C)

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“…The Tris-acetate buffers ranging from pH 5.0 to 9.0 were used a t a final concentration of 0.1 M with respect to acetic acid a t pH5.0 to 6.5 and with respect to Tris-base a t pH 7.0 to 9.0. p2H was determined according to Lumry et al [26] accompanied by an increase in V that was calculated to be about ten-fold. This picture appears to be analogous to that found previously for the conversion of the forward nonenergy-linked reaction into the energy-linked reaction [7], which is accompanied by a decreased dissociation constant for the NADHenzyme complex, i.e.…”

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confidence: 99%

Evidence for a Proton‐Dependent Regulation of Mitochondrial Nicotinamide‐Nucleotide Transhydrogenase

Rydström

1974

European Journal of Biochemistry

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The relationship between energy requirement, [NADf] [NADPH]/[NADH] [NADP]+ ratio and velocity of the nicotinamide-nucleotide transhydrogenase reaction, catalyzed by submitochondrial particles from beef heart, was investigated under conditions where the p H and the energy level were varied.2. At low energy levels, obtained by decreasing the rate of oxidation of respiratory substrates, energy-dependent anilinonaphthalene sulfanote fluorescence is a sensitive tool for estimating the relative energy requirement by the transhydrogenase reaction.3. The velocity of the energy-linked transhydrogenase reaction is directly proportional to the anilinonaphthalene sulfonate fluorescence.4. The energy requirement is related t o the total rate of the transhydrogenase reaction rather than to the degree of activation of the reaction by energy. Under conditions where the energy requirement and the total rate of the transhydrogenase reaction are constant, a decreased pH is accompanied by a decrease in the activation by energy. 5.It is concluded that the kinetic effect and the thermodynamic effect of energy on the transhydrogenase reaction may be separated by varying the pH of the reaction medium. Acidification in the absence of an energy source promotes the conversion of the transhydrogenase into an active form, presumably a protonated species of the enzyme, the kinetic properties of which are similar to those of the energy-activated enzyme. I n the presence of an energy source acidification may replace energization as the means for activation of the transhydrogenase.6. The relationship between the proton-activated and the energy-activated forms of the transhydrogenase with regard to the coupling mechanism is discussed.The energy-linked nicotinamide-nucleotide transhydrogenase reaction catalyzed by submitochondrial particles [l] involves the reduction of NADPf by NADH a t the expense of one high-energy bond, derived from respiration or ATP, per NADPH formed [1-41. This reaction has a V and an apparent equilibrium constant which greatly exceed those of the nonenergy-linked reaction (see [5] for a review). Rydstrom and coworkers [6,7] investigated the steady-state kinetics of the nonenergy-linked and energy-linked reactions, and found that both reactions follow a Theorell-Chance mechanism. They concluded that, mechanisticalIy, the difference between the two reactions can be explained in quantiAbbbreviation. sNAD, thionicotinamide adenine dinucleotide.Enzymes. Alcohol dehydrogenase (EC 1.1.1.1) ; glutathione reductase (EC 1.6.4.2) ; lactate dehydrogenase (EC 1.1.1.27) ; nicotinamide-nucleotide transhydrogenase (EC 1.6.1.1); pyruvate kinase (EC 2.7.1.40); succinate dehydrogenase (EC 1.3.99.1).tative rather than qualitative terms. I n agreement with earlier proposals [3,8], these findings indicated that the same enzyme catalyzes both the nonenergylinked and the energy-linked transhydrogenase reactions.Being a reaction that utilizes either respiratory energy or ATP stoichiometrically, energy-linked transhydrogenation has been a matter ...

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Kinetics of Carboxypeptidase Action. I. Effect of Various Extrinsic Factors on Kinetic Parameters¹

Cited by 230 publications

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Solution Structure of Domain 6 from a Self‐Splicing Group II Intron Ribozyme: A Mg²⁺ Binding Site is Located Close to the Stacked Branch Adenosine

Solution Structure of Domain 6 from a Self‐Splicing Group II Intron Ribozyme: A Mg²⁺ Binding Site is Located Close to the Stacked Branch Adenosine

Metal ion-N7 coordination in a ribozyme branch domain by NMR

Evidence for a Proton‐Dependent Regulation of Mitochondrial Nicotinamide‐Nucleotide Transhydrogenase

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Kinetics of Carboxypeptidase Action. I. Effect of Various Extrinsic Factors on Kinetic Parameters1

Cited by 230 publications

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Solution Structure of Domain 6 from a Self‐Splicing Group II Intron Ribozyme: A Mg2+ Binding Site is Located Close to the Stacked Branch Adenosine

Solution Structure of Domain 6 from a Self‐Splicing Group II Intron Ribozyme: A Mg2+ Binding Site is Located Close to the Stacked Branch Adenosine

Metal ion-N7 coordination in a ribozyme branch domain by NMR

Evidence for a Proton‐Dependent Regulation of Mitochondrial Nicotinamide‐Nucleotide Transhydrogenase

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Kinetics of Carboxypeptidase Action. I. Effect of Various Extrinsic Factors on Kinetic Parameters¹

Solution Structure of Domain 6 from a Self‐Splicing Group II Intron Ribozyme: A Mg²⁺ Binding Site is Located Close to the Stacked Branch Adenosine

Solution Structure of Domain 6 from a Self‐Splicing Group II Intron Ribozyme: A Mg²⁺ Binding Site is Located Close to the Stacked Branch Adenosine