Rate enzyme

The enzyme rate equation with two dissociation relations at equilibrium yields ... 

Lange CC, LP Wackett (1997) Oxidation of aliphatic olefins by toluene dioxygenase enzyme rates and product identification. J Bacterial 179 3858-3865. 

Many parameters can be monitored, for example, free-ion concentrations, membrane potentials, activities of specific enzymes, rate of proton generation, transport of signaling molecules, and gene expression. 

Clinical chemistry Enzyme rate assays, colorimetric assays, end-point assays, immunoassays Upstone, 2000 ... 

Obviously, there must be a limit to the turnover of any enzyme. Rates cannot theoretically go on increasing indefinitely with substrate concentration. In the case of mammalian catalases, the limits appear to lie in the range between a first order rate of 2 x 10 sec and 1x10 sec (36). That is, each heme active site can theoretically decompose between 2 and 10 million molecules of H2O2 per second. As two molecules... 

Fig. 7.22 Relationship between sigma value and enzyme rate for glucuronyl and sulphotransferases indicating the role of nucleophilicity.

There are three basic methods for carrying out alternative substrate inhibition studies. In the first, the investigator seeks to observe numerical changes in the coefficients of the double-reciprocal form of the enzyme rate expression in the presence and absence of the alternative substrate. For some mechanisms, only certain coefficients will be altered. This method requires extremely accurate estimates of the magnitudes of the coefficients and should always be supplemented with other kinetic probes . 

A method for deriving enzyme-rate expressions combining both rapid equilibrium and steady-state procedures first illustrated by Chak With this method, demonstrated by Fromm and Huang, a different rate expression will be obtained depending on which steps are chosen to be in rapid equilibrium and which steps are not. See Enzyme Kinetic Derivations Turnover Number S. Cha (1988) J. Biol. Chem. 243, 820. 

Then, k equals the observed reaction rate divided by the initial reactant concentration i.e., k = Vinitiai/[Ainitiai])-This method is most useful when one has an assay method that is sufficiently sensitive to ensure that only a small fraction, say 3-5%, of the reactant is depleted during the rate measurements. Typically, this is satisfactorily achieved with a UV-visible spectrophotometer, a fluorescence spectrometer, or a radioactively labeled reactant. The initial rate method is extremely convenient, and the preponderance of enzyme rate data has been obtained by initial rate measurements. Finally, one should note that the initial rate method can yield erroneous results if the initial reactant concentration is in doubt. This is not true for the plots of ln ([Ao] - [At]/([Ao] -[Aoo]) versus reaction time because one is considering the fraction of reactant A remaining. 

DERIVATION OF MORE COMPLICATED RATE EQUATIONS. So far, the rate equations that describe one-substrate enzyme systems have been fairly simple, and the usual algebraic manipulations of substitution and/or addition of simultaneous equations have permitted us to obtain the pertinent rate law. When the number of steps increases and especially when there are branched pathways involved, these manual methods become cumbersome, and more systematic procedures are required. The next two sections should allow the reader to develop a working knowledge of effective methods for obtaining multisubstrate enzyme rate expressions. 

ENZYME RATE EQUATIONS (3. Derivation of Isotope Exchange Rate Equations )... 

FROMM S METHOD FOR DERIVING ENZYME RATE EQUATIONS... 

STEP TEN Eliminate any closed loop terms. There are no closed loop terms in the example given above (if there were it would either contain the product kikgkskj or k2kik(,k. Care should be exercised here. Although closed loop terms are rare, not considering them in enzyme rate derivations can lead to incorrect expressions and, thus, inaccurate and erroneous predictions of enzyme rate behavior. A good check is to write out all of the rate-constant products that would constitute a closed loop and then check each enzyme determinant to see if any are present. If so, they are eliminated. Huang s modification of Fromm s systematic approach also addresses this issue of closed loops. 

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