Kinetic and Titration Methods for Determination of Active Site Contents of Enzyme and Catalytic Antibody Preparations

Brocklehurst, Keith; Resmini, Marina; Topham, Christopher M.

doi:10.1006/meth.2001.1176

Cited by 17 publications

(11 citation statements)

References 87 publications

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“…(Equations 1 to 13 are given in the Experimental Procedures below.) The first‐order kinetic constant (called k obs in is obtained by directly fitting data to a single exponential equivalent to [19–21]. The dependence of k obs values on [IN] allows parameters K m ′ and k cat ′ to be determined according to and .…”

Section: Resultsmentioning

confidence: 99%

“…The nonlinear dependence of k obs on IN concentration (Fig. 2A) precludes the possibility that the assay contains a large proportion of denaturated forms of IN that originate from the purification procedure, although this approach cannot rule out the presence of a binding but catalytically inactive form of IN [21]. However, our data show that IN bound in a nonspecific manner is not definitively trapped as most complexes initially formed, even if nonproductive on a short timescale, are potentially active.…”

Section: Discussionmentioning

confidence: 99%

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Kinetic study of the HIV−1 DNA 3′‐end processing

et al. 2006

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Integration of a DNA copy of the human immunodeficiency virus (HIV)-1 RNA genome into the human genome is an essential step in the viral replication cycle. This process is catalysed by a viral protein, integrase (IN). The first key reaction in the overall integration process is the cleavage of a dinucleotide from each 3¢-end of the viral DNA (substrate DNA), a reaction termed 3¢-end processing. In the second step, DNA strand transfer, a pair of processed DNA ends of the same viral DNA is inserted into the host cellular DNA (target DNA). The 3¢-end processing reaction requires a conserved nucleotide sequence at the viral DNA ends, but the second reaction does not absolutely require specific sequences within the host DNA. The 3¢-processing of viral DNA extremities is the first step in the integration process catalysed by human immunodeficiency virus (HIV)-1 integrase (IN). This reaction is relatively inefficient and processed DNAs are usually detected in vitro under conditions of excess enzyme. Despite such experimental conditions, steady-state Michaelis-Menten formalism is often applied to calculate characteristic equilibrium ⁄ kinetic constants of IN. We found that the amount of processed product was not significantly affected under conditions of excess DNA substrate, indicating that IN has a limited turnover for DNA cleavage. Therefore, IN works principally in a singleturnover mode and is intrinsically very slow (single-turnover rate constant ¼ 0.004 min )1 ), suggesting that IN activity is mainly limited at the chemistry step or at a stage that precedes chemistry. Moreover, fluorescence experiments showed that IN-DNA product complexes were very stable over the time-course of the reaction. Binding isotherms of IN to DNA substrate and product also indicate tight binding of IN to the reaction product. Therefore, the slow cleavage rate and limited product release prevent or greatly reduce subsequent turnover. Nevertheless, the time-course of product formation approximates to a straight line for 90 min (apparent initial velocity), but we show that this linear phase is due to the slow single-turnover rate constant and does not indicate steady-state multiple turnover. Finally, our data ruled out the possibility that there were large amounts of inactive proteins or dead-end complexes in the assay. Most of complexes initially formed were active although dramatically slow.Abbreviations HIV, human immunodeficiency virus; IN, integrase; LTR, long terminal repeat; PIC, preintegration complex; r, anisotropy; RSV, Rous sarcoma virus.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Kinetic study of the HIV−1 DNA 3′‐end processing

et al. 2006

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“…Active site titration is a method that can be used to determine the fraction of active enzymes. Protocols are often enzyme-specific, but generally rely on the availability of a strong-binding inhibitor for the enzyme under study [89]. Active site titration has successfully been applied to lipases from Candida antarctica (CALB) after…”

Section: Active Site Titrationmentioning

confidence: 99%

Enzymes immobilized in mesoporous silica: A physical–chemical perspective

Carlsson

Gustafsson

Thörn

et al. 2014

Advances in Colloid and Interface Science

222

167

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“…The sign of ρ therefore should be positive for both cases and not able to distinguish between them. The mechanism shown in Figure 2(b) [ElcB], however, would be Table 1 Kinetic parameters for the non-catalysed (hydroxide ion) and PCA 271-100-catalysed hydrolysis of the substituted arylcarbamates 5a-5d at pH 8.0 and 25 • C The Hammett σ − value (+ 1.27) rather than the standard σ value of + 0.78 is generally used for the 4-nitro group in reactions such as the hydroxide ion reaction (E1cB) characterized by large values of ρ, (> 2) [11,12]; values of k cat were calculated from k cat = 5V /[IgG] (see the text and [16][17][18]). The proficiency constant [25], k cat /(k non-cat · K m ), is related to the specificity constant, k cat /K m , with the intrinsic reactivity of the substrate taken into account.…”

Section: Kinetic Characteristicsmentioning

confidence: 99%

“…After demonstration of adherence of the corrected v versus [S] o data to the Michaelis-Menten equation by the linearity of an [S] o /v versus [S] o plot, the provisional values of the parameters V and K m thus obtained were refined by fitting the Michaelis-Menten equation to the v, [S] o data pairs by weighted non-linear regression using the Sigmaplot 8.0 program. Values of the catalytic rate constant (k cat ) were calculated from k cat = 10 V/2[IgG] = 5 V/[IgG] (10 % of the IgG catalytic and two potential active centres per molecule) to provide lower limits for this parameter[16][17][18].…”

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confidence: 99%

Evidence that the mechanism of antibody-catalysed hydrolysis of arylcarbamates can be determined by the structure of the immunogen used to elicit the catalytic antibody

Boucher

Said

Ostler

et al. 2007

Biochemical Journal

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A kinetically homogeneous anti-phosphate catalytic antibody preparation was shown to catalyse the hydrolysis of a series of O-aryl N-methyl carbamates containing various substituents in the 4-position of the O-phenyl group. The specific nature of the antibody catalysis was demonstrated by the adherence of these reactions to the Michaelis-Menten equation, the complete inhibition by a hapten analogue, and the failure of the antibody to catalyse the hydrolysis of the 2-nitrophenyl analogue of the 4-nitrophenylcarbamate substrate. Hammett sigma-rho analysis suggests that both the non-catalysed and antibody-catalysed reactions proceed by mechanisms in which development of the aryloxyanion of the leaving group is well advanced in the transition state of the rate-determining step. This is probably the ElcB (elimination-addition) mechanism for the non-catalysed reaction, but for the antibody-catalysed reaction might be either ElcB or B(Ac)2 (addition-elimination), in which the elimination of the aryloxy group from the tetrahedral intermediate has become rate-determining. This result provides evidence of the dominance of recognition of phenolate ion character in the phosphate hapten in the elicitation process, and is discussed in connection with data from the literature that suggest a B(Ac)2 mechanism, with rate-determining formation of the tetrahedral intermediate for the hydrolysis of carbamate substrates catalysed by an antibody elicited by a phosphonamidate hapten in which phenolate anion character is minimized. The present paper contributes to the growing awareness that small differences in the structure of haptens can produce large differences in catalytic characteristics.

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Kinetic and Titration Methods for Determination of Active Site Contents of Enzyme and Catalytic Antibody Preparations

Cited by 17 publications

References 87 publications

Kinetic study of the HIV−1 DNA 3′‐end processing

Kinetic study of the HIV−1 DNA 3′‐end processing

Enzymes immobilized in mesoporous silica: A physical–chemical perspective

Evidence that the mechanism of antibody-catalysed hydrolysis of arylcarbamates can be determined by the structure of the immunogen used to elicit the catalytic antibody

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