Direct Estimate of the Specificity Constant: A Possibility or a Fluke? Pre-steady-state Substrate Concentrations and Enabling Mathematical Equations

Udema, Ikechukwu Iloh

doi:10.1101/2023.04.09.536186

Cited by 3 publications

(5 citation statements)

References 48 publications

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Section: Resultsmentioning

confidence: 89%

Higher precision in initial rates may be achievable: A test of a pseudo-statistical method

Ikechukwu Iloh Udema

2023

GSC Biol. Pharm. Sci.

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There has been a concerted effort at establishing the best method for the measurement of initial rates for various purposes, including the calculation of kinetic parameters, the maximum velocity (Vmax), and the Michaelis-Menten constant (KM). The objectives of this research are: 1) to derive equations without KM for the determination of the Vmax in particular and vice versa; 2) to determine the KM and Vmax with other equations other than the Michaelian equation; and 3) to subject the calculated and extrapolated kinetic parameters to pseudo-statistical remediation where necessary as a test of their viability and usefulness. The study was experimental and theoretical. It is supported by the Bernfeld method of enzyme assay. By graphical means, the Vmax and KM values for galactosidase respectively range between 163 and 185 mM/min and between 2.07 and 2.77 mg/L; the range by calculations is 177 and 214 mM/min and 2.45 and 3.311 mg/L, subject to pseudo-statistical remediation. Overall, the ranges of Vmax and KM values for alpha-amylase from both the graphical method and calculation are, respectively, 1.095 to 1.018 mM/min and 18.15 to 20.554 g/L. The equations for the determination of the KM and Vmax, which are respectively invariant with respect to each other, were rederived. The initial rates must not be characterised by a mixture of conditions that validate different quasi-steady-state assumptions if the true KM and Vmax are of interest. The new pseudo-statistical method for the remediation of error in all measurements, if necessary, is viable, useful, and robust.

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Section: Resultsmentioning

confidence: 89%

Higher precision in initial rates may be achievable: A test of a pseudo-statistical method

Ikechukwu Iloh Udema

2023

GSC Biol. Pharm. Sci.

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“…The kinetic parameters of interest are the KM, Vmax, (𝑉 max prss ), and SC; they were determined as described by Eq. ( 9) with respect to the determination of KM where [E0] is = 0.02 g/L and in very recent preprints [1,25] as well as Eq. (25b).…”

Section: Determinations Of Kinetic Parametersmentioning

confidence: 89%

“…Beyond the concern for a definite nomenclature for kinetic constant that seems to be faced with conflicting interpretation and the methodology that can give a definite value without the resort to separate determination of maximum velocity, Vmax, of catalysis and Michaelis-Menten constant, KM, is also the need for ways by which such a kinetic constant (specificity constant, SC) can be determined even under very high concentrations of the substrate. It appears the challenge of methodology has been met as observed in the literature [1], but the contentious issue of nomenclature, that is, the exact name of the ratio,…”

Section: Designmentioning

confidence: 99%

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Linking the Pre-steady-state, Steady-state, and Zero-order kinetic parameters together for Industrial and allied applications

Udema¹

2023

Preprint

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Background: There is an increasing application of enzymes to achieving the industrial goal of the production of biomolecules of different kinds relevant to food, textiles, pharmaceuticals, automobiles, environmental remediation, etc.; there appears not to have been a way of linking pre-zero-order kinetics to zero-order kinetics so as to garner key kinetic parameters at very high industrial concentrations of the substrate. This ought to be a prerequisite for industrial (batch or reactor) design. Objectives: The study is carried out with the objectives of deriving equations that can be explored in relating pre-zero-order (otherwise known as pre-steady-state (prss)) to zero-order kinetic parameters, such as the Michaelis-Menten constant (KM), maximum velocity of catalysis (Vmax), and specificity constant (SC), and ultimately evaluating the equations with experimental data. Methods: The study was theoretical and experimental, backed by the Bernfeld method of generating initial rates. Results and discussion: The Vmax for the higher industrial-type concentration of the enzyme (alpha-amylase) is 7812.5 micromoles/L/min, while the KM is 115.1 g/L. The SC obtained by calculation, either by the new equation (Eq. (25b)) or the ratio Vmax:KM, is 67.88 micromoles L/g min, and surprisingly, as against the literature, the SC obtained by a new graphical method is 275.4 micromoles L/g min using sub-KM values of substrate concentrations. The prss Vmax and KM are 2348.62 ± 479.94 micromoles L/g min and 7.41 ± 1.77 g/L. Conclusion: The need to derive an equation linking prss and the zero-order kinetic parameter is justified by the result obtained. In order to establish consistency, future studies may focus on assays at high concentrations of the enzyme and sub-KM concentrations of the substrate so as to observe a repeat of higher SC.

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“…The outcome of this study notwithstanding, one must bear in mind that if there is no error in all measurements (be it 8 or more replicates for each substrate) under conditions that justify the Michaelian equation and underlying assumptions, there cannot be any need for statistical remediation for generating kinetic parameters; thus, the requirement for statistical soundness and absence of any calculation is out of the question [9]. As opined in a recent preprint report [19], there may be calculations depending on the approach to the solution to any problem of interest. For instance, what has been regarded as the best form of the kinetic parameter, the specificity constant (SC), must be calculated given a single intersection in a reciprocal variant of the direct linear plot by taking the reciprocal of the ratio of KM to Vmax.…”

Section: The Ordinate Y Ismentioning

confidence: 89%

Higher Precision in Initial rates may be achievable: A test of a Pseudo-statistical method

Udema¹

2023

Preprint

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Background: There has been a concerted effort at establishing the best method for the measurement of initial rates for various purposes, including the calculation of kinetic parameters, the maximum velocity (Vmax), and the Michaelis-Menten constant (KM). Objectives: The objectives of this research are: 1) to derive equations without KM for the determination of the Vmax in particular and vice versa; 2) to determine the KM and Vmax with other equations other than the Michaelian equation; and 3) to subject the calculated and extrapolated kinetic parameters to pseudo-statistical remediation where necessary as a test of their viability and usefulness. Methods: The study was experimental and theoretical. It is supported by the Bernfeld method of enzyme assay. Result: By graphical means, the Vmax and KM values for galactosidase respectively range between 163 and 185 microM/min and between 2.07 and 2.77 mg/L; the range by calculations is 177 and 214 micro M/min and 2.45 and 3.311 mg/L, subject to pseudo-statistical remediation. Overall, the ranges of Vmax and KM values for alpha-amylase from both the graphical method and calculation are, respectively, 1.095 to 1.018 mM/min and 18.15 to 20.554 g/L. Conclusion: The equations for the determination of the KM and Vmax, which are respectively invariant with respect to each other, were rederived. The initial rates must not be a mixture of both if the true KM and Vmax are of interest. The new pseudo-statistical method for the remediation of error in all measurements, if necessary, is viable, useful, and robust.

show abstract

Direct Estimate of the Specificity Constant: A Possibility or a Fluke? Pre-steady-state Substrate Concentrations and Enabling Mathematical Equations

Cited by 3 publications

References 48 publications

Higher precision in initial rates may be achievable: A test of a pseudo-statistical method

Higher precision in initial rates may be achievable: A test of a pseudo-statistical method

Linking the Pre-steady-state, Steady-state, and Zero-order kinetic parameters together for Industrial and allied applications

Higher Precision in Initial rates may be achievable: A test of a Pseudo-statistical method

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