Single-turnover experiments

Both mechanisms can also rationalize an increase in due to the production of superoxide/HO2 (18.16), which appears to dominate the flux of partially reduced oxygen species generated by certain biomimetic catalysts [Boulatov et al., 2002 Boulatov, 2004]. It remains to be estabhshed if either of these two mechanisms does indeed operate in simple Fe porph5Tins, for example by carrying out single-turnover experiments similarly to the approach used to study ORR by C5hochrome c oxidase. 

Figure 2b shows the results of a rapid-quench single-turnover experiment performed with EPSP synthase with enzyme in excess over the radiolabeled substrate, PEP. The data show the transient formation and decay of the tetrahedral intermediate, which led to its subsequent isolation and stmcture determination. 

The location of cytochrome C2 in the periplasmic space of purple photosynthetic bacteria has been demonstrated directly by its prompt release following the preparation of sphaeroplasts, and by its accessibility to antibodies in these preparations [220]. Cytochromes c are oxidized in single turnover experiments with a biphasic kinetics (<1 2 and 200-400 /is) this pattern has been interpreted as due to the presence in chromatophores of both cyt. Cj and C2, which are oxidized in series [122]. 

The mechanism by which xanthine oxidase brings about hydroxylation must take into account the fact that water, rather than O2 is the ultimate source of oxygen incorporated into the product. In a single-turnover experiment using H2 0, the radioisotope is not incorporated into the product, whereas, when the enzyme from that experiment is incubated with substrate in unlabelled water, 8- 0-uric acid is produced. It follows that... 

A factor that can influence C02 hydration/dehydration reactivity is the overall coordination number of the zinc center and the coordination mode of a bicarbonate ligand (Fig. 7). It is reasonable to suggest that a unidentate coordinated HCO will be easier to displace, which could influence the rate of the overall hydration reaction. Data discussed below in terms of single turnover experiments supports the notion that bidentate bicarbonate coordination inhibits catalytic C02 hydration. Similarly, bidentate coordination of HCO could be expected to slow the dehydration reaction. Notably, X-ray crystallographic studies of bicarbonate-bound forms of a mutant CA-II, and a Co(II)-substituted form of the enzyme, have revealed both monodentate and bidentate coordination modes for the bicarbonate anion.28,32,45... 

It is important to evaluate the data from the single turnover experiments discussed earlier together with the steady-state turnover data... 

Fig. 11. Suggested catalytic cycle for active MMOH (J. D. Lipscomb version). The cycle, compiled from 31, 48, 50, 53, 56, 63, 64,127-129,132,136,147-150), represents different compounds that have been characterized and some hypothetical compounds (K, R). The rate constants are from single-turnover experiments at 4°C 147).

The simplest vay to measure an isotope effect is the noncompetitive technique, in vhich the rate (kn) with fully protiated substrate ( H labeled), is compared to the rate (kn) at which deuterium labeled substrate ( H labeled) reacts [28]. The label may be in the primary or a secondary position, yielding the primary or secondary KIE, respectively. Steady-state noncompetitive measurements yield the isotope effect on the rate constants or k st/Ku, but suffer from the requirement of both high substrate purity and isotopic enrichment, and from a large uncertainty in the KIE (ca. 5-10%) due to propagated errors. Single-turnover experiments can yield noncompetitive KIEs on the chemical step, but also generally have large uncertainties. Nevertheless, noncompetitive measurements are the only way to obtain KIEs on kcat, which for certain enzymes may be the sole kinetic parameter that reflects the chemical step(s). 

Observed isotope effects will approach intrinsic values when the commitment to catalysis is small (C = 0). Prior to tunneling analyses, earlier single-turnover experiments [81] and steady-state studies [82] had indicated conditions under which Ch would be small or zero. 

Perform single-turnover experiments with enzyme in excess of substrate to examine more closely the conversion of substrates to products at the active site of the enzyme and to look for intermediates. 

In a single-turnover experiment with enzyme in excess, the kinetics of the reaction are different than with substrate in excess. The rate of substrate binding... 

The rate of phosphate binding to EPSP synthase was measured by a single-turnover experiment in the reverse reaction shown in Fig. 10. The experiment was initiated by mixing an excess of enzyme and EPSP with a trace of labeled phosphate (<1 fiM). Under these conditions, the rate of formation of PEP was limited by the rate of phosphate binding to the enzyme-EPSP complex. However, in the absence of unlabeled PEP, the reaction did not go to completion. Successful execution of this experiment required the addition of the unlabeled reaction product (PEP) in order to ensure that the release of radiolabeled PEP was irreversible. In the absence of unlabeled PEP, the reaction came to equilibrium short of complete conversion of radiolabeled phosphate to PEP. The addition of unlabeled PEP pulled the reaction to completion by dilution of the radiolabeled PEP. Computer simulation was required to analyze quantitatively the reaction time course. Conventional data fitting to the time dependence of the reaction gives a rate of approximately 0.035 sec . The simple interpretation would then lead to calculation of a second-order rate constant for phosphate... 

The Schiff base (27) formed in the initial phase of the reaction catalyzed by lumazine synthase can be observed in single-turnover experiments as an optical transient with an absorption maximum at 330nm. A later optical transient with an absorption maximum at 445 nm has been assigned to the product resulting from phosphate elimination. Surprisingly, the ring closure reaction at the end of the reaction sequence appears as the rate-determining step. ... 

Coupled to the folding of the myosin molecule is a change at the active site that results in "trapping" of nucleotide (Cross et al., 1986). The observed rate of phosphate release from single-turnover experiments, <0.0005 sec i, likely reflects product release from the small amount of extended myosin that is in equilibrium with the folded monomer (Cross et al., 1988). The structural basis for trapping is not known. It has been proposed that after MgATP is cleaved at the active site, phosphate leaves via a "backdoor" mechanism (Yount et al., 1995). Perhaps in the folded monomer the glycine-rich P-loop is stabilized in a conformation that prevents phosphate release. 

Co(lII), or Rh(lII), which form inert complexes with nucleotides that exchange ligands on the time scale of days or weeks (especially at low temperatures). It is possible, for example, to separate the A and A isomers of CrATP and use them as substrates in single turnover experiments with various enzymes 23, 24). When the enzyme catalyzes multiple turnovers, the developing circular dichroic (CD) spectrum as one isomer is converted to a product without a CD spectrum can determine the screw-sense specificity. Thus, hexokinase and glycerokinase use the A isomer of CrATP as a substrate, and pyruvate kinase and myokinase (adenylate kinase) use the A isomer (25). The absolute configurations of the ADP and ATP complexes of these metal ions are now known and have been correlated with the CD spectra (26-30). 

The catalytically active state of the radical SAM cluster was first clearly demonstrated via single turnover experiments performed on the PFL-AE. In these experiments, PFL-AE was reduced from the [4Fe S] state to the [4Fe S] state by photoreduction with 5-deazariboflavin by removing the source of illumination, the two specific states of the cluster could be examined for their ability to generate the glycyl radical on PFL in the absence of exogenous reductant. It was found that the quantity of glycyl radical generated on PFL was... 

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