Enzyme kinetics initial rate

A more recent examination of the kinetics of this enzyme by initial rate measurements has included product inhibition patterns and has led to the conclusion that at least under some conditions an ordered bi-bi mechanism applies which involves a ternary complex of enzyme, NAD, and dihydrolipoamide (157). Clear spectral evidence is presented for the existence of a complex between NAD and the oxidized enzyme and this will be discussed in Section III,E. The product inhibition pattern for NAD tended toward that expected for this mechanism only at high NAD concentration. 

Enzymatic assay methods are classified as fixed-time assays, fixed-change assays, or kinetic (initial rate) assays. Kinetic assays continuously monitor concentration as a function of time pseudo-first-order conditions generally apply up to 10% completion of the reaction to allow the initial reaction rate to be determined. If the initial substrate concentration is > 10Km, then the initial rate is directly proportional to enzyme concentration. At low initial substrate concentrations (< 0.1 Km), the initial rate will be directly proportional to initial substrate concentration (cf. Chapter 2). For enzyme quantitation, a plot of initial rate against [E] provides a linear... 

Kinetic measurements11051 have to be carried out to examine the dependence of the reaction rate on the concentrations of all relevant components. As described in a previous chapter, for measuring enzyme kinetics initial reaction rates v0 = j[ ] are determined at optimal reaction conditions, which may be chosen according to the procedure outlined in Sect. 7.3. The initial reaction rates are measured by varying the concentration of only one component and keeping all other concentrations (e. g. of cosubstrates and inhibitors) at a constant level (for an example, see Figs. 7-19 and 7-20). The rate of conversion has to be smaller than 5-10%, essentially to keep all initial concentration values constant. 

Usually initial rates are measured in enzyme kinetics so as to avoid problems arising from kinetic complications such as product inhibition. 

The initial reaction rate (v0) obtained from each substrate concentration was fitted to Michaelis-Menten kinetics using enzyme kinetics. Pro (EKP) Software (ChemSW product,... 

Complex inactivation kinetics caused by enzyme-catalyzed decomposition of epoxide kinetic constants calculated from initial rates of inactivation. Approximate value calculated from half-life in the presence of 50 mAf inhibitor. 

Volume 63. Enzyme Kinetics and Mechanism (Part A Initial Rate and Inhibitor Methods)... 

Both purified laccase as well as the crude enzyme from the WRF Cerrena unicolor were used to convert the dyes in aqueous solution. Biotransformation of the dyes was followed spectrophotometrically and confirmed by high performance liquid chromatography. The results indicate that the decolorization mechanism follows MichaeliseMenten kinetic and that the initial rate of decolorization depends both on the structure of the dye and on the concentration of the dye. Surprisingly, one recalcitrant azo dye (AR 27) was decolorized merely by purified laccase in the absence of any redox mediator [46],... 

Because of the complexity of biological systems, Eq. (1) as the differential form of Michaelis-Menten kinetics is often analyzed using the initial rate method. Due to the restriction of the initial range of conversion, unwanted influences such as reversible product formation, effects due to enzyme inhibition, or side reactions are reduced to a minimum. The major disadvantage of this procedure is that a relatively large number of experiments must be conducted in order to determine the desired rate constants. 

Kinetics of Free and Mao-osoib-Bound eaiymc. The results of the Lineweaver-Burk analysis of the initial rates for free and immobilized enzyme appear in Table I. The increase in the substrate affinity parameter due to some internal mass transfer limitations in the IME as no substrate-matrix interactions were present. The increase... 

H. J. Fromm (1975) Initial Rate Enzyme Kinetics, Springer-Verlag, New York. 

While requiring the availability of competitive inhibitors for each of the substrates, Fromm s use of competitive inhibitors to distinguish multisubstrate enzyme kinetic pathways represents the most powerful initial rate method. See Alternative Substrate Inhibition... 

H. J. Fromm and V. Zewe (1962) J. Biol. Chem. 237, 3027. H. J. Fromm (1975) Initial Rate Enzyme Kinetics, pp. 94-110, Springer-Verlag, New York. 

An initial-rate method introduced by Fromm to discriminate between rival three-substrate enzyme kinetic mechanisms. See Fromm Method for Ternary Systems. 

The velocity of an enzyme-catalyzed reaction can be measured either by a continuous assay or by a stopped-time protocol. Whenever possible, the continuous measurement of a velocity (e.g., the increase or decrease in absorbance vx. time) should be utilized. In stopped-time assays, the investigator must demonstrate that the reaction is completely terminated at the specified point in time and that products are readily and quantitatively separated from substrates. In addition, one must show that the system is under initial rate conditions. Thus, at least three or four different time points should be chosen. Stopped-time assays also require an assay blank (for t = 0). In this blank, typically the quenching conditions are applied prior to the initiation step. Whenever practicable, replicate kinetic analyses should be done, even with continuous assay protocols. See Enzyme Assay Methods Basal Rate... 

The availability of completely pure enzyme affords the opportunity (a) to investigate mechanistic and regulatory properties in the absence of contaminating enzyme activities, and (b) to determine those kinetic parameters requiring an accurate knowledge of the enzyme concentration. Nevertheless, most kinetic studies do not require pure enzyme. What is required in that there be no (or, at least, minimal) contaminating activities that interfere with the initial-rate assay. A contaminating protein may act on the substrate(s), product(s), or effector(s). Hence,... 

A potential limitation encountered when one seeks to characterize the kinetic binding order of certain rapid equilibrium enzyme-catalyzed reactions containing specific abortive complexes. Frieden pointed out that initial rate kinetics alone were limited in the ability to distinguish a rapid equilibrium random Bi Bi mechanism from a rapid equilibrium ordered Bi Bi mechanism if the ordered mechanism could also form the EB and EP abortive complexes. Isotope exchange at equilibrium experiments would also be ineffective. However, such a dilemma would be a problem only for those rapid equilibrium enzymes having fccat values less than 30-50 sec h For those rapid equilibrium systems in which kcat is small, Frieden s dilemma necessitates the use of procedures other than standard initial rate kinetics. 

A linear graphical method for analyzing the initial rate kinetics of enzyme-catalyzed reactions. In the Hanes plot, [A]/v is plotted as a function of [A], where v is the initial rate and [A] is the substrate concentration ". ... 

One of the basic assumptions in kinetic studies of an enzyme-catalyzed reaction is that true initial rates are being measured. In such cases, a plot of the product concentration versus time must yield a straight line. (This behavior is only observed when the substrate is at or near its initial (or, r = 0) concentration. As time increases, product accumulation and substrate depletion will result in a curvature of this progress curve hence, the reaction velocity at these later times would be correspondingly lower.)... 

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