Enzymes single-turnover experiments

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 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... 

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). 

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... 

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). 

Inorganic chiral thiophosphate analysis of fructose bisphosphatase indicates overall inversion of stereochemistry (Domanico et ai, 1986). To distinguish between direct in-line displacement and the intermediacy of an acyl phosphate, a single-turnover experiment was performed. Isotopic equilibration of the putative enzyme-CO 2 with labelled water through multiple turnovers leads to label incorporation. A subsequent single turnover experiment with one equivalent of substrate in unlabelled water must lead to label... 

The proposed mechanism for RDO enzymes has arisen from steady-state kinetic experiments and single turnover experiments carried out by Ballou and Lipscomb, Figure The binding of... 

Catalytic and single-turnover experiments with the R. sphaeroides DMSO reductase, 0-labeled DMSO and l,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane as an oxygen atom acceptor have been used to demonstrate that the enzyme is an oxotransferase. Complementary resonance Raman studies have been interpreted on the basis of a direct mechanism for OAT, with the active site cycling between mono-oxo-Mo(VI) and des-oxo-Mo(IV) forms via a DMSO-bound Mo(IV) intermediate. Both MPT dithiolene groups stay firmly attached to the molybdenum throughout the catalytic cycle. However, EPR and UV/visible spectroscopic evidence has been interpreted on the basis of the species formed upon the addition of DMS to oxidized... 

The method of isotope trapping allows one to detemrine the stickiness of all substrates but the last to combine in an ordered mechanism and of all substrates in a random mechanism (Rose et al, 1974 Rose 1995). This method was developed initially by Rose for the yeast hexokinase reaction, and it is essentially a single turnover experiment in which one detemtines analytically the proportion of an enzyme-substrate complex that reacts to give products, as opposed to dissociating. 

The process of identifying the products of the interaction between the enzyme and alternate substrate depends a great deal on the inhibitor itself. If the compound contains a chromophore or fluorophore, changes in the absorbance or fluorescence spectra with the addition of enzyme can be monitored and used to identify products (Krantz et al., 1990). For multiple product reactions, single turnover experiments can be used to determine relative product distribution. Stoichiometric quantities of enzyme and inhibitor can be incubated for full inhibition, followed by the addition of a rapid irreversible inhibitor of the enzyme, such as an affinity label. This will act as a trap for enzyme as the enzyme-inhibitor complex breaks down. Analysis of the products will determine relative... 

Single turnover experiments can also be used to test whether the sites of oligomeric enzymes are kinetically identical. For instance, lactate dehydrogenase has four active sites per molecule. The following parallel experiments were carried out... 

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