Single turnover reactions

Collman JP, Decreau RA, Yan Y, Yoon J, Solomon El. 2007a. Intramolecular single-turnover reaction in a cytochrome c oxidase model bearing a Tyr244 mimic. J Am Chem Soc 129 5794. 

D. Yields and Rate Constants with Single Turnover Reactions... 

Protein B from M. capsulatus (Bath) not only increases the product yields, but also influences the rate constant for the single turnover reaction of Hred with nitrobenzene (51,67). The pseudo-first-order rate constant increases up to 33-fold when Hred is titrated with protein B. Neither addition of reductase to Hox or Hred, nor addition of protein B and reductase to Hred, could similarly affect the rate constant. These... 

Like O radicals on V and Mo oxides discussed above, Oa exhibits a very high reactivity. At room temperature, it readily oxidizes various organic molecules, including methane. This allows one to conduct single turnover reactions on the catalyst surface, providing in particular the synthesis of phenol according to the... 

In the MMO OB3b system, the values could be used to predict the concentration dependence of the MMOB enhancement on the rate of the multiple turnover reaction. The fit to the experimental data predicts that the maximum rate is attained when a stoichiometric ternary complex (based on active site concentration) is established. Excess MMOB is inhibitory, apparently due to the formation of inactive MMOB-MMOR and MMOB-MMOB complexes, or perhaps binding of MMOB in the MMOR binding site. Cross-linking experiments were used to demonstrate the formation of each of these inhibitory complexes. Component complexes also play a significant role during the single turnover reaction as described below. 

The recent discovery that several radical-SAM enzymes contain an Fe-S cluster in addition to the [4Fe-4S] cluster that is responsible for binding and cleaving SAM has led to proposals for new roles for Fe-S clusters. Spectroscopic and crystallographic studies of BioB have revealed the presence of an additional [2Fe-2S] + cluster that has been shown to act as the immediate sulfur donor for biotin formation from dethiobiotin in a single turnover reaction (equation 5). 

Figure 5 Chemoenzymatic approaches for the production of novel bioactive compounds. In this example, the enzymatic buildup of the linear precursor of daptomycin by its NRPSs (DptA, DptBC, and DptD) is substituted by solid-phase synthesis (a). By using the 4 Ppan transferase Sfp and the CoA-thioester of the linear peptide, the opo-enzyme PCP-TE and be modified, and after trans-esterification cyclized by the TE domain (b). Because the resulting ho/o-enzyme cannot be modified again, this is a single turnover reaction. Another strategy uses thiophenole-esters of the linear peptides to be cyclized (c). When these compounds are used, no PCP domain is necessary. The TE domain is readily acylated, and regiospecific and stereospecific cyclization toward daptomycin or, depending on the linear peptide provided, toward variants thereof occurs. Because the enzyme is not altered in any way after product release, this setup results in a multiple turnover.

Figure 2 Intermediate in the EPSP synthase pathway, (a) The mechanism of the reaction catalyzed by EPSP synthase is shown. The reaction proceeds by an addition-elimination mechanism via a stable tetrahedral intermediate, (b) A single turnover reaction is shown in which 10- xM enzyme was mixed with 1 OO-m-M S3P and 3.5-riM radiolabeled PEP. Analysis by rapid-quench kinetic methods showed the reaction of PEP to form the intermediate, which then decayed to form EPSP in a single turnover. The smooth lines were computed from a complete model by numerical integration of the equations based on a global fit to all available data. Reproduced with permission from Reference 7.

Fig. 3.9. Single turnover reaction sequence. (Reproduced, with permission, from Ref. 66).

Binuclear zinc complexes have been shown to mediate the hydrolysis of the activated peptide model substrate L-leucine- -nitroanilide (LNA, Fig. 32) with the reactions being followed spectroscopically by the formation of /7-nitroaniline ( max 400 nm).165-167 This model substrate has also been used in studies of ApAP.170 The hydrolysis of LNA mediated by the zinc complex [(bomp-)]Zn2(CH3C02)2]BPh4 (Fig. 33a H(bomp) 2,6-bis[bis(2-methoxyethyl)aminomethyl]-4-methylphenol) in water/DMF (6 4) occurs via a reaction that is first-order in complex and substrate, with a second-order rate constant k = 2.3(1) x 10 3M 1 s-1 at 25 °C. At best, a yield of 65% for a single turnover reaction was obtained, indicating that... 

The MMOR transfers electrons from NADH to the oxygen activation site on the iron-oxygen clusters on MMOH. The role of the MMOB is very complex and not yet clarified (31). The electrons from NADH can be replaced by chemical reduction of the two iron-oxygen clusters of MMOH (by, e.g., dithionite) in so-called single-turnover reactions by the MMOH alone (128). MMOH alone can also use the H2O2 reaction (2b) as a replacement for both electrons and oxygen (132) in a so-called peroxide shunt reaction. Both these results clearly demonstrate that the active site is on the MMOH, and that methanol formation does not involve the other two components. 

Following the preparation of the jS-y complex of Cr +ATP, careful chromatography yields four isomers of the complex, two l and two d. Initial studies were performed with each of the purified diastereomers on hexokinase 61). Analysis demonstrated that only one isomer, A, served as a substrate for this enzyme and that the other diastereomer was unreactive. Subsequent studies on other kinases demonstrated varying selectivity for different kinases for the A or A isomers. These studies were performed with either complexes of ADP or ATP. Selectivity was based on potency of inhibition of the various isomers in many cases and the observation of single-turnover reaction in several other cases. If analogy of these structures with those of Mg ADP and Mg ATP complexes holds, individual enzymes have different stereoselectivities for the M-nucleotide structures. A summary of the selectivity of varying M-nucleotide complexes for some enzymes is presented in Table IV 61-65). 

The single-turnover reaction of MMOH ed with O2 has been monitored by time-resolved spectroscopic techniques. Stopped-flow optical spectroscopic and rapid freeze-quench (RFQ)... 

Figure 2. Effect of added coupling protein B on the rate of 4-nitrophenol formation from nitrobenzene in single turnover reactions of Hred dioxygen.

As has been described above and also elsewhere, the role of GTP in EF-Tu- and EF-G-promoted reactions appears to be quite analogous. In both cases, the conformation as well as reactivity of the protein molecules are reversibly and qualitatively altered by the chan of its nucleotide ligands. A single turnover reaction is accomplished utilizing the specific conformation induced by GTP, and the splitting of GTP is required to shift the protein to an alternate conformation. 

The oxidation of alkanes by r-butyl hydroperoxide (TBHP) has been catalysed by titanium alkoxides, producing the corresponding alcohols and ketones. A radical mechanism is proposed in which r-butoxyl radical formed from TBHP and titanium alkoxide initiates the reaction. The evolution of oxygen (from the decomposition of peroxide) and the abstraction of hydrogen from alkane to form alkyl radical occur competitively. A method for the determination of both the primary and secondary KIEs at a reactive centre based on starting-material reactivities allows the determination of the separate KIEs in reactions for which neither product analysis nor absolute rate measurements are applicable. It has been applied to the FeCls-catalysed oxidation of ethylbenzene with TBHP, which exhibits both a primary KIE and a substantial secondary KIE the findings are in accordance with previous mechanistic studies of this reaction. The oxidation of two l-arylazo-2-hydroxynaphthalene-6-sulfonate dyes by peroxy-acids and TBHP catalysed by iron(III) 5,10,15,20-tetra(2,6-dichloro-2-sulfonatophenyl)porphyrin [Fe(ni)P] is a two-step process. In single turnover reactions, dye and Fe(in)P compete for the initially formed OFe(IV)P+ in a fast reaction and OFe(IV)P is produced the peroxy acid dye stoichiometry is 1 1. This is followed by a slow phase with 2 1 peroxy acid dye stoichiometry [equivalent to a... 

Figure 10. Proton inventory of cytochrome P450cam second electron transfer and oxygen activation. Rates were normalized relative to the rates of second electron transfer through product release measured in H2O. All data points represent at least three independent determinations of stopped-flow spectrophotometric experiments between dioxygen complexed P450cam (2 iM) and reduced Pd (20 pM) with 2.5 mM metyrapone to stop reactions following single turnover. Reactions were maintained at 8T in D2O/ H2O buffer mixtures listed as mole fraction D2O (n). Data taken from [101].

EPR spectroscopy in combination with stopped-flow absorption and rapid freeze-quench techniques has been employed (i) to probe flie catalytically relevant oxidation state(s) of MIOX and (ii) to investigate the reaction between MIOX, substrate, and O2. While most other oxygen-activating binuclear non-heme iron enzymes are catalytically active in their fully reduced form, MIOX exhibits a raflier different behavior. In single-turnover reactions of the diferrous recombinant Mus... 

For the purpose of the present discussion the term transient kinetics is applied to the time course of a reaction from the moment when enzyme and substrate are mixed, t=0, until either a steady state or equilibrium is established. The difference between the kinetic problems discussed in section 3.3 and in the present section is, respectively, the presence of catalytic as distinct from catalytic concentrations of enzyme. Here we are concerned with the stoichiometry of enzyme states. Transient kinetic experiments with enzymes can be divided into two types. The first of these (multiple turnover) is carried out under the condition that the initial concentrations of substrate and enzyme are Cs(0) Ce(0) and c it) can, therefore, be regarded as constant throughout the course of the reaction until a steady state is attained. Alternatively, in a single turnover reaction, when Cs(0)reaction intermediates is observed until the overall process is essentially complete. These two possibilities will be illustrated with specific examples. In connection with a discussion of the approach to the steady state, in section 3.3 it was emphasized that, at t = 0, the concentrations of the intermediates, enzyme-substrate and enzyme-product complexes, are zero and, therefore, the rate of product formation is also zero. Under the experimental conditions used for steady state rate measurements and for enzyme assays, the first few seconds after the initiation of a reaction are ignored. However, when the experimental techniques and interpretation discussed below are used, events during the first few milliseconds of a reaction can be analysed and provide important information. With suitable monitors it is possible to follow the formation and decay of enzyme complexes with substrates and... 

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