Cleavage reactions, single-turnover

For combination in a twin ribozyme both units should specifically interact with a unique substrate sequence. To this end we changed the substrate sequence in the single units and determined the kinetic constants of the cleavage reaction before constructing the twin ribozymes. All single catalytic modules under single turnover conditions had rate constants between 0.2 and 0.5 min-1. 

Schiff base, or quinoidal intermediates do not appreciably affect the rate of the a reaction.90113 (2) L-serine and amino acids such as O-methyl-L-serine that form ES III do stimulate the a reaction.113 (3) The rate of indole-3-glycerol phosphate turnover is roughly correlated with the rate of formation of ES III for each of the amino acids.90-113 (4) The kinetics of the lag in cleavage of indole-3-glycerol phosphate and synthesis of L-tryptophan under single turnover conditions correspond to the rate of ES III formation... 

Rather surprisingly, the hydration of the imidazole carbon and the cleavage of the first carbon—nitrogen bond are relatively rapid reactions. The rate-determining step is located late in the reaction trajectory. It is therefore possible to distinguish at least four optical transients under single turnover conditions. ... 

After five cycles of selection and ampHfication, a population of single-stranded DNAs was enriched that catalyzed the Pb +-dependent cleavage at the ribose residue. This intramolecular cleavage activity was transformed into an inter-molecular reaction by separating the 38-nucleotide long catalytic domain from the 21-mer substrate which was cleaved specifically and with high turnover rates. Remarkably, the deoxyribozyme can perform well only with the special DNA/RNA chimeric oHgonucleotide substrate and cannot cleave a pure RNA substrate of the same sequence. 

As was noted in Scheme 12, distonic radical cations obtained from cyclopropane bond cleavages add oxygen rapidly, producing products with two CO bonds. So do some alkene radical cations. Addition of O2 to an alkene radical cation is formally a nucleophilic attack by the single alkene n electron on O2, and oxidizes both carbon atoms (an alkene radical cations has formally two -f carbons, and the adduct a 1+ and an oxygen-bound carbon). The oxygenation of the radical cation of bia-damantylidine (96) leads to dioxetanes such as 98 in chain reactions (see Scheme 21) [110]. The reactions may be initiated electrochemically or photochemically, but tris(o,p-dibromophenyl)amine hexafluoroantimonate, 97, is a superior catalyst for the dark reaction of certain tetraalkylalkenes, with turnovers up to ca. 800 at... 

The model for a—j3 intersubunit communication indicates that it is the formation of the aminoacrylate species that leads to activation of the a reaction. When both serine and IGP are added simultaneously to the enzyme in a single enzyme turnover experiment, there is a lag in the cleavage of IGP that is a function of the reaction of serine to form the aminoacrylate species. Accordingly, amino acids other than serine that can undergo dehydration to form the aminoacrylate such as cysteine should serve as alternate substrates but should lead to a longer lag for the a subunit activation as determined by transient kinetic analysis. Cysteine does... 

Substrate specificity of the purified indoleamine 2,3-dioxygenase from rabbit intestine was examined spectrophotometrically at 24°C. The spectra of the reaction products in either the absence or presence of formamidase were compared with those of authentic compounds. A single enzyme protein catalyzed the oxygenative ring cleavage of d- and L-tryptophan, 5-hydroxy-D- and -L-tryptophan, tryptamine, and serotonin (10). The maximal turnover number was obtained with L-tryptophan (99 mol min -mor of enzyme at 24°C), and the lowest value was with 5-hydroxy-L-tryp-tophan (20 p.Af). A marked substrate inhibition is observed by the L isomers of tryptophan and 5-hydroxytryptophan above 0.2 and 0.06 mM, at pH 6.6, respectively. The compounds including skatole, indole, in-doleacetic acid, 5-hydroxyindoleacetic acid, N-acetyltryptophan, melatonin, and a-methyl-DL-tryptophan, are all inert as substrate. 

Hydrolytic cleavage of membrane NAPE by a stimulus-activated PLD activity yields, in a single-step reaction, saturated and unsaturated NAEs. In fact, the NAEs recovered after stimulation of neurons in primary culture closely correspond to those expected from the structure of neuronal NAPE. This is an important piece of evidence in favor of a role of NAPE as NAE precursor but not the only one. Other supportive evidences include the ability of homogenates of neurons and other tissues (e.g., testis) to hydrolyze radiolabeled NAPE forming NAEs and the demonstration that NAPE turnover accompanies NAE formation in stimulated neurons. 

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