Kinetic analysis substrate

While many enzymes have a single substrate, many others have two—and sometimes more than two—substrates and products. The fundamental principles discussed above, while illustrated for single-substrate enzymes, apply also to multisubstrate enzymes. The mathematical expressions used to evaluate multisubstrate reactions are, however, complex. While detailed kinetic analysis of multisubstrate reactions exceeds the scope of this chapter, two-substrate, two-product reactions (termed Bi-Bi reactions) are considered below. 

We first consider the simple example of an uncatalyzed Diels-Alder reaction shown in Scheme 50.2 in order to demonstrate the use of different excess experiments. The Diels-Alder reaction is known to exhibit second overall order kinetics, as shown in eq. (4). We demonstrate with this known case how reaction progress kinetic analysis may be used to extract the reaction orders in both substrate concentrations, [5] and [6]. ... 

Amejdki-Chab, N., Costentin, J., and Bonnet, J. J., Kinetic analysis of the chloride dependence of the neuronal uptake of dopamine and effect of anions on the ability of substrates to compete with the binding of the dopamine uptake inhibitor GBR 12783, J. Neurochem., 58, 793, 1992. 

Measurement of blue and green fluorescence of NADH and FAD in living tissues Quantitative fluorescent cytochemistry Using permeable fluorogenic substrates of enzymes, specific inhibitors, and kinetic analysis... 

Using specific fluorescent transport substrates, inhibitors and kinetic analysis Using fluorescent xenobiotics or fluorescent analogue of xenobiotics Using special fluorescent xenobiotics or fluorescent analogues of xenobiotics Vital tests with acridine orange or neutral red Metachromatic fluorescence of intercalated or bound acridine orange, 590/530 nm microfluorometry... 

The rate law for two diastereomeric catalyst-substrate complexes -symmetric ligands) resulting from Michaelis-Menten kinetics (Eq. (11)) has already been utilized by Halpern et al. for the kinetic analysis of hydrogenations according to Scheme 10.2, and corresponds to Eq. (3) of this study. 

The results of the kinetic analysis for the investigated systems are summarized in Table 10.2, the substrate concentration used being the same for all trials. In the case of methyl- and cyclohexyl-substituted ligands the Michaelis constant is smaller than the initial substrate concentration of [S]o=0.06666 mol L-1 (Table 10.2). However, a description of the hydrogenations with other catalyst ligands as first-order reactions shows that in each of these cases the Michaelis constant must be much greater than the experimentally chosen substrate concentration. 

Respiration inhibition kinetics analysis (RIKA) involves the measurement of the effect of toxicants on the kinetics of biogenic substrate (e.g., butyric acid) removal by activated sludge microorganisms. The kinetic parameters studied are max> the maximum specific substrate removal rate (determined indirectly by measuring the maximum respiration rate), and Ks, the half-saturation coefficient [19]. The procedure consists of measuring with a respirometer the Monod kinetic parameters, Vinax and Ks, in the absence and in the presence of various concentrations of the inhibitory compound. 

Traditionally, HAT activity is measured with a discontinuous radioactive filterbinding assay, which uses pH]acetyl-CoA as a histone acetyltransferase substrate [46]. The transfer of [ H]acetyl-groups to the histone substrate by histone acetyltransferases is detected by liquid scintillation counting of pHjacetylated histones, which are retained on a phosphocellulose disk. Due to its discontinuous character, this assay is technically problematic and not ideal for kinetic analysis. Hence, other assays that work with radiolabeled acetyl-CoA have been described that are suitable for a higher throughput. These work with streptavidin-covered beads [47] or a variant of the SPA with microtiter plates that contain a scintillant (FlashPlates) [48]. But as all these protocols are based on radioactively labeled substrates, they apparently show the same disadvantages that were described for the radioactive HDAC assay protocols. Therefore, nonradioactive assays have been developed to study histone acetyltransferase activity. 

Competitive, 249, 123, 146, 190 [partial, 249, 124 progress curve equations for, 249, 176, 180 for three-substrate systems, 249, 133, 136] competitive-uncompetitive, 249, 138 concave-up hyperbolic, 249, 143 dead-end, 249, 124 [for bireactant kinetic mechanism determination, 249, 130-133 definition of kinetic constants, 249, 220-221 effects on enzyme progress curves, nonlinear regression analysis, 249, 71-72 inhibition constant evaluation, 249, 134-135 kinetic analysis with, 249, 123-143 one-substrate systems, 249, 124-126 unireactant systems, theory,... 

A procedure to simplify the experimental method in the kinetic analysis of three-substrate, enzyme-catalyzed reactions ". In this method, the concentration of one substrate is varied while the other two substrates are kept in a constant ratio and in which the individual concentrations of these two substrates are in the neighborhood of their respective Michaelis constants. The experi-... 

A systematic kinetic analysis of the binding site of an enzyme with the subunits of a polymeric substrate, in-... 

With the exception of a recent bisubstrate kinetic analysis of bilirubin UDP-glucuronyltransferase (P5), saturation with either one of the substrates was investigated at some rather arbitrarily fixed concentration of the other substrate. The results, therefore, have to be interpreted with caution. 

Kinetics of O-Methylaiion. The steady state kinetic analysis of these enzymes (41,42) was consistent with a sequential ordered reaction mechanism, in which 5-adenosyl-L-methionine and 5-adenosyl-L-homocysteine were leading reaction partners and included an abortive EQB complex. Furthermore, all the methyltransferases studied exhibited competitive patterns between 5-adenosyl-L-methionine and its product, whereas the other patterns were either noncompetitive or uncompetitive. Whereas the 6-methylating enzyme was severely inhibited by its respective flavonoid substrate at concentrations close to Km, the other enzymes were less affected. The low inhibition constants of 5-adenosyl-L-homocysteine (Table I) suggests that earlier enzymes of the pathway may regulate the rate of synthesis of the final products. 

ESI-MS has been used for the quantification of a number of substrates and products of enzymatic reactions [56,57]. Hsieh et al. report the use of ion spray mass spectrometry (a technical variation of electrospray ionization) coupled to HPLC for the kinetic analysis of enzymatic reactions in real time [58]. The hydrolysis of dinucleotides with bovine pancreatic ribonuclease A and the hydrolysis of lactose with 3-galactosidase were monitored and the resulting data were used for the estimation of and v x of these reactions. Another field of application of electrospray mass spectrometry is the screening of combinatorial libraries for potent inhibitors [31,59]. 

From bisubstrate, kinetic analysis with a transferase from hen oviduct that, under the conditions of the assay, formed only GlcNAc-PP-Dol, it followed that both dolichol phosphate and UDP-GlcNAe have to he bound to the enzyme before release of the product occurs.52 However, the fact that only partially purified preparations have thus far been obtained (the preparations may also still be contaminated with substrates and product), together with experimental difficulties in handling both the substrate dolichol phosphate (which, furthermore, is not one compound, see the earlier discussion) and the unstable enzyme (enveloped in micelles of detergent), make difficult a sensible interpretation and comparison of the kinetic parameters detenuined for the different enzvme-preparations. The solubilized enzymes catalyzing reactions 1,2, and 3 have in common their alkaline pH optima and dependence on Mg2+ or Mn2+ ions. The latter fact makes (ethylenedinitrilo)tetraacetic acid (EDTA) a reversible inhibitor of enzyme activity and an important experimental tool. 

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